A man revealed to be Jacques Saunière is being pursued by a mysterious hooded character known as Silas through the Grand Gallery in the Louvre in Paris. (Silas is later revealed to be a member of the ultra-conservative Catholic sect called Opus Dei that wishes to annihilate a secret society called the Priory of Sion, who is sworn to protect the Holy Grail. Proof that the Grail exists would ultimately reveal a devastating secret that would undermine the fundamental teachings of the Roman Catholic Church.) Silas demands the location of the Priory's clef de voûte or "keystone." Under threat of death, Saunière finally confesses that the keystone is kept in the sacristy of Church of Saint-Sulpice, "beneath the Rose." Silas thanks him, and then shoots him in the stomach.
Meanwhile, American symbologist Robert Langdon (Tom Hanks), who is in Paris as an AUP guest lecturer on symbols and the sacred feminine, is contacted by the French police, and summoned to the Louvre to view the crime scene. He discovers that the dying Saunière had created an intricate display using his own body and blood. Captain Bezu Fache (Jean Reno) asks him for his interpretation of the puzzling scene. Langdon determines that the pentacle drawn on the stomach and the way the body was posed was similar to Leonardo da Vinci's drawing of the "Vitruvian Man." A cryptic message, written in blood, is found next to the body. It begins with the Fibonacci sequence of numbers, but not in the correct order.
Silas calls a mysterious man known as "The Teacher", revealing that he has killed all four protectors of the keystone and that all confirmed the same location. He dons a metal cilice on his thigh and proceeds to flagellate himself with a whip for the sins of murder. Facilitated by Bishop Manuel Aringarosa, Silas then travels to Saint-Sulpice and is admitted by an elderly nun; left alone, he excavates beneath the floor of the church to find a stone saying only JOB 38:11. He confronts the nun, who quotes the passage: "Hitherto shalt thou come, but no further." Realizing that he has been deceived, Silas is enraged and kills the nun.
Sophie Neveu (Audrey Tautou), a cryptologist with the French police, enters the Louvre as well and slips Langdon a message which leads him to the men's room. There, Sophie meets him and tells him that he is being tracked, a GPS tracking dot has been (unknown by him) slipped into his jacket and that he is a primary suspect in the murder case because of a line of text found by the corpse ("P.S. find Robert Langdon"). Sophie however, believes that Saunière, who is revealed to be her grandfather, wanted to pass a hidden message on to her, and that he had wanted to bring Langdon into the equation so that he could help her crack the code.
Having bought some time by removing the tracking device, the pair begin exploring the Louvre based on their deciphering the message written in blood. It turns out that the part of the message that reads O Draconian devil Oh lame saint is actually an anagram, which, when the letters are re-arranged, spells Leonardo da Vinci and The Mona Lisa. Searching near that painting, they find another encrypted message, So dark the con of man, that turns out to be an anagram for [i/Madonna of the Rocks, another da Vinci painting found at the Louvre. Near that second painting, the pair find a key with a Fleur-de-lis.
Pursued by the French police and cut off from the United States Embassy, the pair escape to the Bois de Boulogne where Langdon closely inspects the key. He notices an inscription on the side - an address. The address directs them to the Depository Bank of Zurich where the key is used for a safety deposit box.
In the bank, they find Saunière's deposit box and open it using the 10 digit Fibonacci numbers in order (1123581321). Inside the box, they find a rosewood container, which contains a cryptex: a cylindrical container with five alphabetical dials which must be arranged in the correct sequence to spell out a 5-letter code word, in order to open and access the parchment message inside. Using force to open the cryptex would break a vial of vinegar inside, which would dissolve the parchment and destroy the message.
Unfortunately, the police are called by a security guard and they are forced to leave. The bank manager, Andre Vernet, assists them in escaping by taking them as passengers in an armoured van to escape the routine checks of the police. In the back of the truck Langdon and Neveu have a lengthy discussion about the cryptex and Neveu says that her grandfather often played games with her involving cryptexes. Langdon says that the cryptex might hold valuable information or another clue about what they are trying to discover. Eventually, they come to a sudden stop and Vernet forces them at gunpoint to give him the cryptex. Langdon tricks Vernet and disarms him and he and Sophie escape with the cryptex in their hands.
Langdon suggests that they visit his friend, Leigh Teabing (Ian McKellen), for assistance to opening the cryptex. Leigh Teabing turns out to be an enthusiastic seeker of the Holy Grail, which he believes is not actually a cup ("sangreal" as in Holy Grail) but instead Mary Magdalene ("sang real" as in Royal Bloodline), who was driven away because Jesus's followers didn't want to follow a woman after their leader was killed. Mary was pregnant at the time, and Teabing tells Sophie that a secret society--the Priory of Sion along with its military arm, the Knights Templar--was formed to protect the descendants of Jesus. Jacques Saunière was believed to be a part of this society and Teabing suspects that he was training Sophie to join it also. Silas, meanwhile, breaks into Teabing's mansion and attempts to steal the cryptex. Teabing uses his cane to knock Silas out and they escape again, taking the butler, Remy Jean, and Silas with them.
The group escapes in Teabing's plane, following the next clue to London. This clue was found underneath the rose carved on the wooden box containing the cryptex. The message, which had to be read using a mirror, refers to "a knight a pope interred." Having barely slipped away from the London police (who were tipped by the French that fugitives were on board Teabing's private jet), Langdon and Sophie forego a visit to the library and, instead, use a fellow bus passenger's smartphone to do an internet search on keywords mentioned in the clue. The search results made them realize that the pope referred to Alexander Pope, who wrote the epitaph of Sir Isaac Newton (the knight). This leads them to Newton's tomb at Westminster Abbey.
The small pyramid beneath the Inverse Glass Pyramid removed from underneath, revealing that there is no chamber as shown in the film's closing scene. It is revealed that Remy Jean is actually a follower of The Teacher as well, however he is killed by the mysterious man after freeing Silas. Silas is attacked by the police and, in the ensuing gunfire, accidentally shoots Bishop Manuel Aringarosa. In his grief, Silas dies in police-assisted suicide and Aringarosa is taken to the hospital, as well as being arrested by Fache for betraying him.
As Langdon gets closer to solving the mystery, he is betrayed by Teabing, who is revealed to be The Teacher. Teabing explained that he wanted to find Mary Magdalene's remains to prove he was correct about the Holy Grail and threatens to shoot Sophie if Langdon does not crack the code. Langdon responds by throwing the cryptex into the air. Teabing catches it, but drops it, and it hits the ground. The vial of vinegar breaks and apparently spreads onto the document, destroying it.
After Teabing is arrested, it is revealed that Langdon had cracked the code and removed the clue from the cryptex before throwing it. The code word had something to do with an orb located somewhere on the tomb, but Langdon later realized that, "There was every orb conceivable on that tomb except one: The orb which fell from the heavens and inspired Newtons life's work. Work that incurred the wrath of the church until his dying day. A-P-P-L-E. Apple."
The parchment inside the cryptex had the following message: The Holy Grail 'neath ancient Rosslyn waits / The blade and chalice watch o'er her gates / Adorned by masters loving art she lies / As she rests beneath the starry skies. Using the clue, they travel to Rosslyn Chapel in Scotland where Magdalene's remains had previously been hidden. In a secret, underground chamber, Langdon notices how the clue precisely describes this chamber, but the center of the room is empty, as if Magdalene's tomb has been removed.
At the chapel, they meet other members of the secret organization that protected Mary Magdalene. It is revealed that Sophie is actually Magdalene's descendant and therefore is the current living descendant of Jesus Christ. They vow to keep her safe. As Sophie wonders if her secret should be revealed to the world despite the disappearance of Magdalene's remains (whereby DNA testing could have proven that Sophie is indeed a descendant), Langdon philosophizes that, if given a chance, would you rather destroy faith...or renew it? As Langdon and Sophie part ways, Sophie sets foot in a pond in an attempt to walk on water. Unsuccessful, she jokes, "Maybe I'll do better with the wine."
Back in Paris, Langdon accidentally cuts himself while shaving and the line of blood on the sink reminds him of the Rose Line. He follows the Rose Line and, realizing that the clue from the cryptex also fits this new location, he determines that the location of the Holy Grail is buried under the pyramid in the Louvre. Langdon then kneels above Mary Magdalene's tomb as the Templar Knights did before him.