Peritoneal carcinomatosis of colorectal cancer is common and associated with poor prognosis, which poses a serious challenge and satisfactory treatments are urgently needed. Hyperthermic CO2 pneumoperitoneum (HT-CO2) is a new strategy. This study was designed to determine the potential of HT-CO2 against colorectal cancer cells. Based on an in vitro HT-CO2 study model, the anti-tumor efficacy of HT-CO2 (42-44 degrees C for 2-4 h) on human colon cancer COLO 205 cells was evaluated and the mechanisms of actions were analyzed. We found that HT-CO2 (43-44 degrees C for 2-4 h) significantly decreased cell viability as determined by WST-8 assay, and the cytotoxicity was attributable to HT-CO2-induced hyperthermia and extracellular acidification. Apoptosis was the major form of cell killing as demonstrated by Annexin-V/PI flow cytometry and morphological analysis (Hoechst/PI fluorescence microscopy and transmission electron microscopy). Further Western blot analysis and flow cytometric analysis of mitochondrial membrane potential showed that Bax-associated mitochondrial apoptotic pathway played critical role in the induction of apoptosis. We conclude that HT-CO2 has significant cytotoxic effect on colon cancer cells through induction of Bax-associated mitochondrial apoptosis, and the cytocidal effect is attributable to HT-CO2-induced hyperthermia and extracellular acidifications. Our data suggest that HT-CO2 may serve as a potential candidate for treating and/or preventing peritoneal carcinomatosis of colorectal cancer and further investigations both in vitro as well as in vivo in animal models are needed.