Sertoli cells (SCs) function as “nurse cells,” which play crucial roles in supporting spermatogenesis through establishing a unique and essential environment in the male reproductive tract. Given the important roles of SCs in male fertility, this study was designed to evaluate the effect of diosgenin, an aglycone of the steroidal saponin, on TM4 cell proliferation and to elucidate the possible mechanisms. We showed that diosgenin increased the proliferation of TM4 cell and primary SCs in a time- and concentration-dependent manner. Diosgenin increased cyclins D1 and E as well as CDK4/6 and CDK2 expression but inhibited P27 expression, with no significant alterations of cyclin B and cdc2 (cell division cycle 2), resulting in cell-cycle G₁/S transition. Diosgenin significantly inhibited apoptosis, as reflected by decreased percentage of TUNEL-positive cells; decreased expression of Bax (Bcl-2-associated X protein), AIF (apoptosis-inducing factor), and cleaved caspases 3 and 9; and increased expression of Bcl-2 (B-cell lymphoma 2). Diosgenin induced an immediate and transient plasma membrane translocation of ESR1 and ESR2 from the nucleus, which was inhibited by the antiestrogen ICI 182 780 and PP2, an inhibitor of SRC. Moreover, ICI 182 780 and PP2 significantly inhibited diosgenin-induced cell-cycle transition and inhibition of apoptosis. Activation of extracellular regulated protein kinase (ERK)/Akt signaling was also involved in diosgenin-induced TM4 cell proliferation, which was SRC- and ESR-dependent. Furthermore, diosgenin induced late activation of nuclear ESR transcriptional activity, which in turn directly regulated cell cycle and apoptosis-related factors, such as cyclin D and Bcl-2. Taken together, the results show that diosgenin activated SRC-ESR translocation-ERK/Akt-ESR transcriptional activity, leading to cell-cycle transition and inhibition of apoptosis and thus final cell proliferation. These findings may better our understanding of the pharmacological actions of diosgenin and advance therapeutic approaches to male infertility.