Dietary isoflavones, a group of secondary plant compounds that exhibit phytoestrogenic properties, are primarily found in soy. Prunetin, a representative isoflavone, was recently found to affect cell signaling in cultured cells; however, in vivo effects remain elusive. In this study, the model organism Drosophila melanogaster was used to investigate the effects of prunetin in vivo with respect to lifespan, locomotion, body composition, metabolism, and gut health. Adult flies were chronically administered a prunetin-supplemented diet. Prunetin improved median survival by 3 d, and climbing activity increased by 54% in males. In comparison with the females, male flies exhibited lower climbing activity, which was reversed by prunetin intake. Furthermore, prunetin-fed males exhibited increased expression of the longevity gene Sirtuin 1 (Sir2) (22%), as well as elevated AMPK activation (51%) and triglyceride levels (29%), whereas glucose levels decreased (36%). As females are long-lived compared with their male counterparts and exhibit higher triglyceride levels, prunetin apparently "feminizes" male flies via its estrogenicity. We conclude that the lifespan-prolonging effects of prunetin in the male fruit fly depend on changes in AMPK-regulated energy homeostasis via male "feminization." Collectively, we identified prunetin as a plant bioactive compound capable of improving health status and survival in male D. melanogaster.-Piegholdt, S., Rimbach, G., Wagner, A. E. The phytoestrogen prunetin affects body composition and improves fitness and lifespan in male Drosophila melanogaster. FASEB J. 30, 948-958 (2016). www.fasebj.orgDiet plays an important role in health and in the prevention of chronic diseases (1). The traditional Asian diet is rich in fruits, vegetables, and legumes, including soy. Soy is the most important dietary source of isoflavones, and prunetin is a representative of the isoflavone group that exhibits potent bioactivity (2). Although it has been demonstrated in cultured cells in vitro that prunetin may affect cell signaling, little is known about its bioactivity in vivo. We investigated whether prunetin affects health and lifespan in the model organism Drosophila melanogaster, given that inflammation, stress response, barrier function, and metabolism have been described as crucial determinants of longevity. In this context, D. melanogaster is an appropriate model for investigating the effects of plant bioactives on metabolism, inflammation, and aging, because genes that affect common biologic processes and molecular functions are evolutionarily conserved. Drosophila exhibits orthologs of most mammalian genes. Furthermore, numerous Drosophila protein sequences are similar to those of mammals (3). In addition, the fruit fly possesses a complex, dynamic gut that is similar in structure and organization to the mammalian gut (4). Insect immune function has much in common with the innate immune response of mammals, and the fruit fly is an accepted model for investigating innate immunity [reviewed i...