Germ cell apoptosis is crucial for spermatogenesis and can be triggered by various stimuli, including intratesticular hormone deprivation. This study proposes a role for insulin-like growth factor binding protein-3 (IGFBP-3) in male germ cell apoptosis. Groups of adult Sprague-Dawley male rats received one of the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of the gonadotropin-releasing hormone antagonist (GnRH-A), acyline, on day 1 and a daily IT injection of saline; (iii) daily IT injection of IGFBP-3; and (iv) a GnRH-A injection on day 1 and a daily IT injection of IGFBP-3. Germ cell apoptosis increased significantly after IGFBP-3 or GnRH-A treatment which was further enhanced by the combined treatment. After co-immunoprecipitation with BAX antibody, IGFBP-3 association with BAX was demonstrated in total and mitochondrial fractions but not in the cytosol of testis extracts. BAX-associated IGFBP-3 expression was increased in mitochondria after treatment compared with control, which was confirmed by an IGFBP-3 enzyme-linked immunosorbent assay. Dot blot studies further validated the BAX-IGFBP-3 binding in vitro. IGFBP-3 as well as BAX induced release of cytochrome c and DIABLO from isolated testicular mitochondria in vitro. IGFBP-3, when combined with an ineffective dose of BAX, triggered release of these proteins from isolated mitochondria at a 4-fold lower dose than IGFBP-3 alone. Our data demonstrate that the IGFBP-3 and BAX interaction activates germ cell apoptosis via the mitochondria-dependent pathway. This represents a novel pathway regulating germ call homeostasis that may have significance for male fertility and testicular disease.