Low oxygen caused by a decreasing blood supply is known to induce various responses of cells, including apoptosis. The present study was conducted to examine whether low-oxygen conditions (hypoxia) induce luteal cell apoptosis in cattle. Bovine midluteal cells incubated under hypoxia (3% O₂) showed significantly more cell death than did those incubated under normoxia (20% O₂) at 24 and 48 h of culture, and had significantly lower progesterone (P4) levels starting at 8 h. Characteristic features of apoptosis, such as shrunken nuclei and DNA fragmentation, were observed in cells cultured under hypoxia for 48 h. Hypoxia increased the mRNA expressions of BNIP3 and caspase 3 at 24 and 48 h of culture. Hypoxia had no significant effect on the expressions of BCL2 and BAX mRNA. Hypoxia also increased BNIP3 protein, and activated capsase-3. Treatment of P4 attenuated cell death, caspase-3 mRNA expression, and caspase-3 activity under hypoxia. Overall results of the present study indicate that hypoxia induces luteal cell apoptosis by enhancing the expression of proapoptotic protein, BNIP3, and by activating caspase-3, and that the induction of apoptosis by hypoxia is partially caused by a decrease in P4 production. Because hypoxia suppresses P4 synthesis in bovine luteal cells, we suggest that oxygen deficiency caused by a decreasing blood supply in bovine corpus luteum is one of the major factors contributing to both functional and structural luteolysis.