The present study explored the inhibitory effect of hyperthermic CO2 pneumoperitoneum on the proliferation and migration of colon cancer cells, and its mechanism. Colon cancer cell line SW-480 was sealed into a urine collection bag to simulate pneumoperitoneum with 100% CO2 under a pressure of 14 mmHg. The growth and morphology of cells were observed under a microscope, the inhibition on cell proliferation was measured using WST-8 test, cell apoptosis and the cell cycle were monitored using fluorescence-activated cell sorting analysis, the migration of cells was tested using the scratch assay, and the expression of HSP-70, caspase-3, hypoxia-inducible factor-1α (HIF-1α) and matrix metalloproteinase-9 (MMP-9) proteins and genes was investigated using western blotting and reverse transcription polymerase chain reaction. Compared with the control group, there was no significant difference in the CO2 group (P>0.05), while the apoptosis and necrosis rates in the hyperthermo-CO2 group was significantly increased (P<0.05). Compared with the control group, the number of cells at G0/G1 phase significantly increased and the number of cells at S phase significantly decreased in the hyperthermo-CO2 group (P<0.05), indicating that hyperthermo-CO2 could arrest the cell cycle. It was suggested by the results of the scratch assay that cell migration ability enhanced in the CO2 group, but decreased in the hyperthermo-CO2 group compared with the control. CO2 pneumoperitoneum promoted cell migration by upregulating HIF-1α and MMP-9 expression. However, the CO2 pneumoperitoneum with hyperthermia enhanced apoptosis and inhibited migration by upregulating the expression of HSP-70, HIF-1α and caspase-3, but downregulating the expression of MMP-9.