Artist Thomas J. Wright painted all of the paintings used to introduce each story.

Conceived as an updating of "The Twilight Zone" concept, Serling reportedly began planning the series soon after The Twilight Zone (1959) was cancelled in 1964.

John Astin appeared in three separate episodes of this show. During each episode, his character was killed, and during two episodes, his character found himself in Hell. He also directed three episodes of the show.

Two segments, and possibly a third, were directed by Steven Spielberg. According to the book, "Rod Serling's Night Gallery: An After Hours Tour", Spielberg was scheduled to direct the 1971 vignette "A Matter of Semantics" starring Cesar Romero. Those involved with the production are unclear in their memory as to whether Spielberg directed the piece, which was ultimately credited to Jack Laird. At least one actor involved in the two-minute mini-episode recalls a director who more closely fits Spielberg's description than Laird's. Beginning with the second season, and despite Rod Serling's objections, the producers began to insert brief one to three minute "blackout comedy" sketches in between main segments of some episodes, usually when an episode was running short. The merits of these brief vignettes remain controversial among this show's fans to this day.

Sculptors Logan Elston and Phil Vanderlei did all of this show's sculptures.

One year before the debut of Kung Fu (1972), David Carradine and Radames Pera, who each played the "Kung Fu" character of Kwai Chang Caine at different ages throughout that series, appeared in the same episode of this show, though in different segments. Carradine appeared in season two, episode sixteen, "Phantom Farmhouse", and Pera appeared in season two, episode seventeen, "Silent Snow, Secret Snow".

Episodes from the hour long The Sixth Sense (1972), starring Gary Collins, were edited down into half-hours, and added to this show's syndication package. Studios find it easier to sell programs in syndication when they have more episodes in the package, as local stations know audiences are more likely to grow tired of a program if the same episodes are repeated over and over again within a short period of time. Adding the edited The Sixth Sense (1972) programs made syndicating the program easier.

The syndicated version of the series took individual segments from the original hour long program and worked them into half-hour episodes. This sometimes meant editing out footage from original episodes that ran longer than a half an hour, or adding footage to those that originally were shorter than half an hour.

Rod Serling had originally conceived of a show like this one when he was still working on The Twilight Zone (1959). He had originally wanted to change the stories to be shown during the final season from fantasy to horror (the genre he preferred), but CBS adamantly refused to agree to it. Unfortunately for Serling, on this show, he did not have the same kind of control over the program like he did on The Twilight Zone (1959), as he was just the host and occasional story contributor. Serling frequently clashed with the show's producer over the quality of stories shown on the program.

Four-In-One (1970) rotated four separate shows: this show, McCloud (1970), San Francisco International Airport (1970), and The Psychiatrist (1970). The last two programs were cancelled after that season; McCloud (1970) became part of the new Mystery Movie series (along with Columbo (1971), and McMillan & Wife (1971)) and this show was given its own time slot.

The set used by Rod Serling to introduce each story contained paintings and sculptures. Only paintings were used, however, to illustrate a story's title.