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The story begins after closing arguments have been presented in a murder case, as the judge is giving his instructions to the jury. According to American law (both then and now), the verdict (whether guilty or not guilty) must be unanimous. A non-unanimous verdict results in a hung jury, which in turn forces a retrial. The question they are deciding is whether the defendant, a young teenaged boy from the city slum, murdered his father. The jury is further instructed that a guilty verdict will be accompanied by a mandatory death sentence — the electric chair. The jury of twelve move to the jury room, where they begin to become acquainted with each others' personalities and discuss the case.

The plot of the film revolves around their difficulty in reaching a unanimous verdict due, in some cases, to the jurors' prejudices. Juror #8 dissents in the initial voting, stating that the evidence presented is circumstantial and the boy deserves a fair deliberation, upon which he starts questioning the accuracy and reliability of the only two witnesses to the murder, the fact that the knife used in the murder is not as unique as assumed (he produces an identical one from his pocket), and the overall shady circumstances.

His most fierce opponents - Jurors 3, 4, and 10 - claim that the boy's alibi is botched, since he does not remember any detail from the movies he watched at the theatre the night of the murder and he has sufficient motivation to kill his father. His lack of memory, however, is excused by panic attack; also, one of the witnesses is accused of wanting attention whilst the other might have "witnessed" the murder without her glasses on. As the deliberation goes on, the jurors go on to vote not guilty - in order, Jurors 9, 5, 11, 2, 6, 7, 12, 1, 4, 10, and finally 3. In a secret ballot, Juror 9 is the first to support Juror 8, not necessarily believing the accused is not guilty but feeling Juror 8 deserves the opportunity for...