Despite evidence from several bird, fish and mammal species that listeners can discriminate between the vocalizations of familiar and unfamiliar adult conspecifics, direct links between discriminatory abilities and fitness benefits have been difficult to identify. In free-ranging populations of African lions (Panthera leo), females with cubs face a substantial threat from one particular category of unfamiliar individuals: infanticidal males. Here we use playback experiments to demonstrate that females with cubs can distinguish immediately between roars from their own resident males (the fathers of the cubs) and those from unfamiliar, potentially infanticidal males. Although they remain relaxed when played roars from resident males, they immediately become agitated on hearing unfamiliar males and retreat rapidly with their cubs if the latter have reached about 4.5 months of age. These responses are not simply a function of the roarers being unfamiliar, for when played the roars of unfamiliar females, females with cubs consistently approach the loudspeaker. Furthermore, females often move toward the cubs in response to playbacks of unfamiliar males but not in response to playbacks of unfamiliar females or resident males. Our results suggest how females with cubs might, by quickly detecting and categorizing unfamiliar intruders within their territory, protect their cubs from infanticidal males and expel intruding females. Distinguishing between individuals on the basis of their vocal characteristics could therefore confer direct fitness benefits on discriminating lionesses.