Males of many vertebrate species aggressively defend their reproductive interests by monopolizing females, and the ‘challenge hypothesis’ predicts that testosterone levels in reproductive contexts rise to facilitate males' competitive behaviours necessary for meeting social challenges. The hypothesis is successful in explaining patterns of testosterone secretion in many avian species, but remains comparatively unexplored in mammals. ‘Circulating plasma testosterone levels (T)’ were studied in relation to harem maintenance in grey-headed flying-foxes, Pteropus poliocephalus . In this species, harems provide mating opportunities and so a male's ability to maintain a harem is likely to correlate with his fitness. We hypothesized that if T reflect a male's ability to withstand challenges from competitors, then T should be linked to successful harem maintenance. To test this, we temporarily removed males from their territories prior to and during the short mating period, recording their harem sizes both before removal and after reintroduction. Most males successfully reclaimed their territory and a harem, but during the mating period, males with higher T had harems closer to their original size, and males with lower T suffered reduction in harem size. Our findings highlight the role of T in harem maintenance in a major mammalian taxon with complex forms of social organization.