Section One, a clandestine anti-terrorist organization, fakes the death of a jailed, convicted murderer and, believing her twin assets of beauty and ability to kill will make her a valuable new operative, trains her in the fighting skills necessary to succeed in her new job. The new operative, code-named 'Josephine', proves to be somewhat less ruthless than planned, however, as she had been falsely convicted and never murdered anyone.

In the original Luc Besson film (and in the American remake Point of No Return (also released as The Assassin), also released by Warner Bros.), Nikita is a drug-addicted juvenile delinquent who kills a police officer in cold blood during an attempted robbery of a pharmacy. She is later arrested and sentenced to life imprisonment in Nikita, and to death by lethal injection in Point of No Return, upon which she was secretly drugged by the government, faking her death. Nikita is then "recruited" by a secret government organization and transformed into a highly skilled assassin who cannot be traced.

The television series differs from the film versions in one fundamental aspect: Nikita (Peta Wilson) is innocent. She is not a killer, nor a drug user, just a homeless young woman in the wrong place at the wrong time. Section One—an elite, top-secret counter-terrorism organization—sets Nikita up to be accused of murdering a police officer and sentenced to life in prison where she supposedly commits suicide and is brought into Section One. Because Nikita will be killed (or "cancelled") if she fails to comply, she is forced to carry out the organization's ruthless methods of fighting terrorism, while attempting to keep her moral integrity intact. This personal struggle becomes the primary conflict of the series. A key scene in the two theatrical films involves Nikita's first assignment—to murder a VIP in a crowded restaurant. Although the Nikita of the television series eventually does become, by necessity, a ruthless killer, in the television version of this first mission she avoids killing the VIP (whereas the film versions of the character completes the assignment). However, despite the machinations of others in power around her, Nikita retains her compassion and humanity.

Over time, Nikita's secretive and risky romantic involvement with her trainer, the mysterious Michael Samuelle (Roy Dupuis), will become another source of conflict, and the series' most significant relationship. Just before the conclusion of the series, Nikita also learns the truth of why she was recruited into Section One.