There is clear evidence that insulin and insulin-like growth factor I (IGF-I) are crucial for the normal metabolism and development of Sertoli cells. However, the mechanisms of insulin regulatory signaling remain unknown in these cells, especially during the immature period. The aim of this study was to investigate the electrophysiological effects of insulin and the effects of insulin and IGF-I on calcium uptake, amino acid, and glucose transport in whole seminiferous tubules from 12-day-old rats, as well as the involvement of PI3K/Akt signaling pathway in these effects. Insulin produces a depolarizing effect on the membrane potential of Sertoli cells in seminiferous tubules within 180 s. This effect was nullified by verapamil, an L-type voltage-dependent calcium channel blocker, therefore demonstrating a calcium-dependent depolarizing effect. Both insulin and IGF-I stimulate calcium uptake, amino acid, and glucose transport in whole testes from 12-day-old rats. These stimulatory effects of insulin and IGF-I on calcium uptake and amino acid and glucose transport on testicular tissue were nullified by wortmannin, which demonstrates the involvement of the PI3K/Akt signaling pathway in these hormonal effects.