Hypoxia has been reported to be an important factor leading to male infertility, and it has been reported that hypoxia can induce the apoptosis of mouse spermatogenic cells. Sirtuin 3 (SIRT3) has been reported to promote the degradation of hypoxia-inducible factor 1α (HIF-1α), and thus, we hypothesized that SIRT3 may influence hypoxia-induced apoptosis of spermatogonia. In this study, we overexpressed or inhibited SIRT3 in mouse type B spermatogonia GC-2 cells and then subjected the cells to hypoxia or normoxia, before examining hypoxia-responsive gene expression and cell viability. We report that SIRT3 stabilizes hypoxia-inducible factor 1α (HIF-1α) and activates its downstream target gene expression in GC-2 cells. We also show that the SIRT3 inhibitor 3-TYP suppresses HIF-1α target gene expression and alleviates hypoxia-induced apoptosis of GC-2 cells. Our study reveals the critical role and underlying mechanisms of SIRT3 in hypoxia-induced apoptosis of mouse type B spermatogonia GC-2 cells.