"Gunslinger Girl" is set in modern-day Italy and revolves around the Social Welfare Agency, a covert government organization that employs cybernetically enhanced young girls as assassins. These girls, referred to as "fratello," are paired with adult male handlers, known as "padrone," who train and oversee their missions. The girls are former victims of severe trauma, rescued from near-death situations and subjected to cybernetic enhancements and psychological conditioning to erase their pasts, rendering them emotionally detached yet fiercely loyal to their handlers.

The series primarily focuses on the relationship dynamics between the girls and their handlers, exploring themes of loyalty, dependency, and the ethical implications of using children as weapons. Henrietta, one of the central characters, is paired with Jose, a compassionate handler who struggles with the morality of his role. Their bond is complex, blending paternal care with professional duty. Other key pairs include Rico and Jean, whose relationship is more pragmatic, and Triela and Hilshire, which highlights the handler's protective instincts.

The narrative unfolds through episodic missions, each revealing more about the girls' pasts and the psychological toll of their conditioning. The missions often involve neutralizing political targets, terrorists, or rogue elements threatening national security. As the series progresses, the girls' humanity begins to resurface, challenging their programmed obedience and raising questions about their autonomy and future.

The overarching plot also introduces rival factions, such as the Five Republics Faction, a terrorist group opposing the Italian government. These external threats create tension and drive the action, while internal conflicts within the Social Welfare Agency further complicate the narrative. The series balances its action-driven plot with introspective moments, delving into the emotional and psychological struggles of its characters.

"Gunslinger Girl" is notable for its somber tone and exploration of moral ambiguity, presenting a world where the lines between right and wrong are often blurred. The series does not shy away from depicting the harsh realities of its premise, making it a thought-provoking and emotionally resonant work within the anime genre.