Oscar-nominated actor Mako was announced as the voice of Splinter at San Diego Comic Con the day before he died. Fortunately, he had finished the majority of his acting.

While Leonardo, Raphael, and Donatello all have brown eyes, Michelangelo has blue eyes. According to writer and director Kevin Munroe, this was done to make Michelangelo seem a bit more innocent and younger than the other turtles.

According to writer and director Kevin Munroe on the DVD commentary, the little song that Splinter sings when he enters the kitchen is an old Japanese lullaby that Mako ad-libbed for the scene.

Final theatrical movie of Mako (Splinter).

During Raphael's finishing lines, the phrase "we strike hard and fade away into the night" was used. That was also the ending line in the first TMNT comic.

Karai's first movie appearance. In her original Mirage comic incarnation, Karai was completely unrelated to the Shredder and is actually a member of the Foot Clan's Council of Five, higher rank than the Shredder in the Foot Clan's global hierarchy. It is presumed Karai has the same status in this movie as she is not referred to as the adopted daughter of Shredder as she was in Teenage Mutant Ninja Turtles (2003).

Some of the thirteen monsters are based on actual "monsters" from popular myth. The first one that appears in the unfinished building is based on the Yeti, the next one to be captured is obviously the Cyclops, the one that eats a pigeon on a building is clearly modelled on a gargoyle, and the one in the diner brings to mind the Jersey Devil.

This movie was dedicated to the memory of Mako (Master Splinter).

During the fight between the Nightwatcher and the monster in the diner, at one point, a refrigerator falls on top of the monster and the music cuts out and the monster is heard making a whimpering noise. This was actually a mistake in the soundtrack, but when the filmmakers saw it, they couldn't stop laughing, and left it in the movie.

As revealed in the director's commentary on the DVD one of the early drafts of the script had Raphael briefly dying, and Donatello using Winter's technology to bring him back to life. This idea was turned down by Peter Laird, who disliked the idea of having one of the turtles dying.

TMNT 2 would have loosely adapted the Turtles' thirteen-part comic book saga "City At War". Michelangelo feels like the "odd man out" amongst his brothers, not being taken seriously. With the best of intentions, the character runs away and joins the Foot Clan, donning a black bandanna. Meanwhile, the Turtles travel to Japan and cross paths with Karai in an adventure resulting in the return of their archnemesis, Shredder. TMNT 3 would have featured the Triceratons as well as the Technodrome's arrival from Dimension X, with Donatello being a catalyst for these events. Kevin Munroe wanted Michael Clarke Duncan to voice the Triceraton's leader, Commander Mozar. He even had poster designs already in mind for TMNT 2 ("a wet city street, by a manhole cover, Mikey's orange bandanna lying on the ground") and its sequel ("the Technodrome with the Turtles very small in the foreground").

The previous movies in the franchise were all live-action. This is the first movie in the franchise in fourteen years. Chronologically, this movie takes place after the first three movies.

It was the number one movie in the U.S. on its opening weekend, bringing in $25.45 million.

Jim Cummings is the only Teenage Mutant Ninja Turtles actor to make it into this movie since his first TMNT appearance in Teenage Mutant Ninja Turtles (1987), which he played Leatherhead, Genghis Frog, and one of the occasional voice alternates for Shredder.

Specific dialogue makes references to the previous live-action movies. In the battle with the Foot Clan, Casey Jones says, "Two minutes for high sticking!" which was one of his lines from his introduction in Teenage Mutant Ninja Turtles (1990). The last line of the movie, spoken by Raphael is, "Man, I love being a turtle!" which has been spoken in each of the previous movies.

The movie's lead track "Shell Shock" by Gym Class Heroes originated as a freestyle rap performed at a concert they performed at.

Until Teenage Mutant Ninja Turtles (2014) this was the movie with the largest budget in the franchise.

Splinter's appearance is generally consistent with the previous movies (in which he is depicted as having brown fur), although in this movie, he wears a proper karate gi, as opposed to dirty rags from the previous movies. His right ear is also no longer cut as it was in previous movies. Writer and director Kevin Munroe jokingly responded that Splinter had an otoplasty.

All of the Generals' names are loosely translated from Spanish: Aguila (means "Eagle"), Gato (means "Cat"), Serpiente (means "Serpent"), and Mono (means "Monkey").

The idea of April O'Neil knowing ninjitsu skills was taken from Teenage Mutant Ninja Turtles (2003) with her receiving some training from Master Splinter, whereas the idea of her wearing a yellow jumpsuit was a nod to Teenage Mutant Ninja Turtles (1987) with her being a reporter wearing a yellow jumpsuit with pockets.

According to James Arnold Taylor (the voice of Leonardo) several characters had already been recorded using established voice artists before the Weinstein Company became involved with the film, at which point the characters were re-voiced by celebrities such as Sarah Michelle Gellar (April O'Neil), Chris Evans (Casey Jones), and Patrick Stewart (Winters).

Despite this movie taking place in New York City, only one real-life New York City landmark was shown, the Brooklyn Bridge. Most of the cityscape in the movie is fictional.

The first entirely animated Ninja Turtle movie.

Kevin Munroe, in his opinion, believes he would probably rather re-cast Splinter, instead of having somebody who sounds like Mako, because he doesn't think anybody can really match Mako.

It is the first Teenage Mutant Ninja Turtles movie made with computer graphics imagery (CGI) by Imagi Animation Studios.

John Woo was supposedly set to direct, but moved on to other projects.

During the first Nightwatcher scene, when the criminals are fleeing, one of the signs on the shops in the background spells out "monro" in hiragana, a Japanese writing system, which is most likely related to writer and director Kevin Munroe.

One of the arcades at the Turtle's lair features the character Zeus from Imagi's animated television series, Zentrix (2001).

One sequel idea that had already been nixed by Peter Laird had the Turtles go back in time to explore Mayan culture, just as an evil alien race also arrives via teleportation. According to Kevin Munroe, Laird's response was, "Eh, we did it in 1992", a reference to Teenage Mutant Ninja Turtles III (1993).

This is the only incarnation in the franchise, out of all the television series and movies (not including Ninja Turtles: The Next Mutation (1997)) to not have the name "Teenage Mutant Ninja Turtles", instead using the initials, which fans usually use to shorten the name.

Greg Baldwin replaced Mako in other projects along with this movie, including the final seasons of Avatar: The Last Airbender (2005) and Samurai Jack (2001).

Splinter use Japanese two times in this movie. The first time is after Leonardo and Raphaels fight at the breakfast table when Splinter walks in to the scene singing in Japanese. One line from his humming/singing that is clearly audible, and sung two times is "Asahi ga noboru" which means "The morning sun rises". The second time he uses Japanese is during the final fight where he holds Winters back, he says "chotto matte", which means "wait a moment".

Michelangelo is voiced by Mikey Kelley. With Mikey being Michelangelo's shorter name, this marks the first time where a turtle has been voiced by a voice actor with the same name.

If the monster that attacked the diner is indeed based on the Jersey Devil as suggested, then it is rather appropriate that Kevin Smith provided the voice of the diner's chef, since he is an avid and loyal New Jersey Devils hockey fan.

First Teenage Mutant Ninja Turtles film to be presented in the 2.35:1 aspect ratio. All of the previous movies were presented in the 1.85:1 aspect ratio.

Marco Beltrami composed a score for the movie, but the score was rejected by Warner execs about three and a half weeks before the movie was released. Klaus Badelt was hired to rewrite the score and compose it at the 7th hour. however, still remain in the finished film.

Warner Bros.' fifth theatrically released computer-animated film after The Polar Express (2004), The Reef (2006), The Ant Bully (2006), and Happy Feet (2006).

The seventh computer-animated film to be produced at 2.35:1 after A Bug's Life (1998), The Polar Express (2004), The Incredibles (2004), Cars (2006), Monster House (2006), and Happy Feet (2006).

This movie is considered by some fans as a loose continuation of the original trilogy, ignoring the events of the television series Ninja Turtles: The Next Mutation (1997), but is really set in its own universe. Towards the end of the movie, several items are shown that reference the live-action movies. These include: The Shredder's helmet and staff, a Foot Ninja Mask, the canister of ooze that mutated the turtles and Splinter (labelled TCRI instead of TGRI, though a crack is strategically placed to work either way), the Time Sceptre, Walker's hat, Lord Norinaga's helmet, and pieces of the suits of armor worn by the Turtles in Teenage Mutant Ninja Turtles III (1993).

The scene where Casey and Raphael are being attacked, Casey says "And I thought insurance salesmen were pushy!" This line was also used in Teenage Mutant Ninja Turtles (1990) by Michaelangelo when the Foot Clan was invading April's apartment.