Abstract: B16 Melanoma cells were inoculated into the BALB/C mice to establish melanoma-bearing models. The antitumor effect of Tween-80 in combination with hyperthermia 41°C was studied. We observed the changes of the mortality of tumor-bearing mice, the tumor growth curves, activities of serum tumor necrosis factor (sTNF) and the level of serum sialic acid (sSA) in tumor-bearing mice. The number of pulmonary metastatic tumor foci from blood flow was also detected. The results showed that combined with Tween-80, hyperthermia 41°C could dramatically suppress the growth of the melanoma in the feet of mice, survive the tumor-bearing mice and decrease the number of pulmonary metastatic tumor foci but no significant effects were observed by treatment with Tween-80 or hyperthermia at 41°C alone. The activities of sTNF and the level of sSA of the melanoma-bearing mice kept at higher levels than those of normal BALB/C mice. Tween-80 combined with heating 41°C significantly decreased the activities of sTNF and increased the level of sSA that decreased 10 weeks later with the tumor regression. These results demonstrate that Tween-80 may make hyperthermia exert effective antitumor effect below the critical temperature and increase the safety of hyperthermia in treatment. It is one of the most ideal synergist with hyperthermia. The changes of sTNF and sSA suggest that the synergetic effect could involve in facilitating the exposure of tumor antigen and activation of immune system.