To investigate the effect of wt p53 gene transfection on the sensitivity of hyperthermia in C6 glioma. The recombinant eukaryotic expressive vector pCMV-p53 plasmids were extracted, identified and transmitted to C6 glioma cells by stable transfection. The biological characteristics were compared among C6/p53 (+) cells, thermo-treated C6/p53 (+) cells, thermo-treated C6 cells, thermotolerance C6 cells and control C6 cells. Transplanted glioma was treated by laser interstitial thermotherapy (LITT) in tumor-bearing nude mice. The wt p53 gene segments were extracted and correctly identified by restriction enzyme (Hind and EcoR). The optimal transfection concentration of plasmid to liposome was 1:6, neo gene expressed stably in C6 cells transfected with positive and blank plasmid. Thermotolerance C6 cell growth was normal in vitro, growth of C6/p53 (+) cells was inhibited. Growth of hyperthermia treated C6 cells was inhibited significantly 12 h after thermal treatment, cell proliferation activity of C6/p53 (+) cells was suppressed significantly 6 h after hyperthermia treatment. Apoptosis was observed in C6/p53 (+), thermo-treat C6 and thermo-treat C6/p53 (+) cells. The apoptosis ratio of hyperthermia treated C6/p53(+) cells was increased by 30 folds compared with that of control groups. Study in nude mouse model demonstrated lower positive inoculation rate in groups of C6/p53 (+) cells (57 %), hyperthermia treated C6 cells (75 %) and hyperthermia treated C6/p53(+) cells (20 %). C6/p53(+) LITT group showed the highest antitumor effect, compared with C6 LITT group(P<0.05). However, thermotolerance C6 LITT group had the lowest effect. Transfection of wt p53 gene can increase the sensitivity of hyperthermia in glioma cells.