Studies have shown that 2,5-hexanedione (2,5-HD) is the main active metabolite of n-hexane in the human body. The toxicity of n-hexane and 2,5-hexanedione has been extensively researched, but toxicity to the reproductive system, especially the impact on female reproductive function, has been less frequently reported. In this study, we exposed human ovarian granulosa cells to 0, 16, 64, and 256 μM 2,5-HD in vitro for 24 h. Through hematoxylin-eosin (HE) staining, Hoechst 33342 staining, transmission electron microscopy, and flow cytometry using FITC-Annexin V/PI double staining, 2,5-HD was demonstrated to cause significant apoptosis of human ovarian granulosa cells in a dose-dependent manner. As part of our continuing studies, we investigated the underlying apoptosis mechanism of human ovarian granulosa cells exposed to 0, 16, 64, and 256 μM 2,5-HD in vitro for 24 h. Real-time quantitative PCR and Western blot analysis were used to detect changes in the expression of the apoptosis-related BCL-2 family (BCL-2, BAX) and CASPASE family (CASPASE-3) with increasing 2,5-HD concentration. The results showed that with increasing 2,5-HD doses, the expression of BCL-2 decreased. However, a marked dose-dependent increase in the expression of BAX and active CASPASE-3 (p17) was observed in human ovarian granulosa cells. These results suggest that the mechanisms of 2,5-HD causing increased apoptosis in human ovarian granulosa cells might be through BCL-2, BAX, and CASPASE-3 signaling pathways.