This study was designed to investigate the proliferation inhibition and apoptosis-promoting effect under hyperthermia and chemotherapy treatment, at cellular level. Human gastric cancer cell line SGC-7901 was cultivated with 5-fluorouracil at different temperatures. Cell proliferation and apoptosis were determined, and expression of Bcl-2 and HSP70 was measured at different treatments. Cell survival rates and inhibition rates in chemotherapy group, thermotherapy group, and thermo-chemotherapy group were drastically lower than the control group (P<0.05). For tumor cells in the thermo-chemotherapy group, survival rates and inhibition rates at three different temperatures were all significantly lower than those in chemotherapy group and thermotherapy group (P<0.05). 5-Fluorouracil induced apoptosis of SGC-7901 cells with a strong temperature dependence, which increased gradually with increase in temperature. At 37°C and 43°C there were significant differences between the thermotherapy group and chemotherapy group and between the thermo-chemotherapy group and thermotherapy group (P<0.01). The expression of Bcl-2 was downregulated and HSP70 was upregulated, with increase in temperature in all groups. Cell apoptosis was not significant at 46°C (P>0.05), which was probably due to thermotolerance caused by HSP70 accumulation. These results suggested that hyperthermia combined with 5-fluorouracil had a synergistic effect in promoting apoptosis and enhancing thermotolerance in gastric cancer cell line SGC-7901.