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Gunslinger Girl's setting is modern day Italy. It follows the lives of several girls who are part of the Social Welfare Agency (referred to mostly as "The Agency"), a counter terrorism group posing as a government facility that helps in the rehabilitation of the physically injured.

The girls are usually those that have been left for dead, after suffering excessive amounts of physical and psychological damage. The majority of their bodies are replaced with synthetic tissue and go through a brainwashing procedure, which is termed "conditioning" by the Agency. The conditioning makes them forget about their past and makes them obey the orders of their trainers without a doubt, as it creates a bonding effect (less like love, more like adoration/worship). The girl and trainer pair (all trainers, who are also called handlers, have been male so far) are dubbed 'fratello', which means 'brother' in Italian.

They are much stronger than normal humans, more tolerant of pain, will unfailingly obey orders, and if necessary, die for their handlers.