Insulin is essential for the regulation of glucose homeostasis. Insulin dysfunction occurs in several pathologies, such as diabetes mellitus, which is associated with fertility problems. Somatic Sertoli cells (SCs) not only metabolize glucose to lactate, which is the central energy source used by developing germ cells, but also determine the germ cell population size. If a deregulation in SCs apoptosis occurs, it will affect germ cells, compromising spermatogenesis. As SCs apoptotic signaling is a hormonally regulated process, we hypothesized that the lack of insulin could lead to alterations in apoptotic signaling. Therefore, we examined the effect of insulin deprivation on several markers of apoptotic signaling in cultured rat SCs. We determined mRNA and protein expression of apoptotic markers as well as caspase-3 activity. SCs cultured in insulin deprivation demonstrated a significant decrease on mRNA levels of p53, Bax, caspase-9, and caspase-3 followed by a significant increase of Bax and decrease of caspase-9 protein levels relatively to the control. Caspase-3 activity was also decreased in SCs cultured in insulin deprivation conditions. Our results show that insulin deprivation decreases caspase-dependent apoptotic signaling in cultured rat SCs evidencing a possible mechanism by which lack of insulin can affect spermatogenesis and fertility.