The present in vitro Sertoli cell-enriched culture study was aimed to understand the precise role of FSH and testosterone either alone or in combination in regulation of Sertoli cell nursing function such as lactate production, which in turn is utilized by the germ cells as an energy substrate. The stimulatory effect of FSH on lactate production by Sertoli cell-enriched culture was significantly enhanced when FSH and dihydrotestosterone were supplemented in the medium either simultaneously or DHT was added 12 h later to FSH (0 h FSH+12 h DHT). On the contrary, DHT alone or added 12 h prior to FSH (0 h DHT+12 h FSH) in the incubation medium, significantly reduced the lactate secretion by Sertoli cells as compared to control or FSH alone/FSH+DHT simultaneously/0 h FSH+12 h DHT, respectively. This provides the conclusive evidence that androgen has additive effect on nursing function of Sertoli cell-enriched clusters only when they are in an activated state in response to gonadotropin.