The effects and potential molecular mechanisms underlying carbon dioxide (CO(2)) pneumoperitoneum on gastric cancer cell apoptosis are not fully understood. In this study, we assessed the effects of CO(2) pneumoperitoneum on the apoptosis of MKN-45 gastric cancer cells. Additionally, we investigated the role of HIF-1alpha in CO(2) pneumoperitoneum-induced apoptosis of gastric cancer cells. MKN-45 cells were cultured in CO(2) or air pneumoperitoneum at 0, 12 and 15 mmHg pressures for 4 h. We observed a change in cells morphology and increasing apoptotic ratios in MKN-45 cells when they were put into a 15 mmHg CO(2) pneumoperitoneum environment. However, there was no significant difference between the 0, 12 mmHg CO(2) pneumoperitoneum and the control groups. Exposure to 15 mmHg CO(2) pneumoperitoneum significantly enhanced the expression levels of HIF-1alpha and Bax, while it attenuated Bcl-2 expression levels. When we inhibited HIF-1alpha by small interfering RNA (siRNA), we found that the apoptotic ratio of MKN-45 cells decreased in 15 mmHg CO(2) pneumoperitoneum. This treatment markedly elevated Bcl-2 levels and decreased Bax expression. These data suggest that CO(2) pneumoperitoneum may accelerate the apoptosis of MKN-45 cells at higher pressures. HIF-1alpha is a crucial factor that affects gastric cancer cell apoptosis by downregulating the Bcl-2/Bax ratio.