Abstract: Objective The study objective was to investigate the effect of hyperthermia combined with rhTNF on cell cycle and F-actin of TNFR1 in over-expressed glioma, as well as invasiveness in vitro. Methods C6 cell Line of over-expressed TNFR1 (C6/TNFR1) was constructed., The mRNA and protein of TNFR1 were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively, and the cell cycle and cell proliferation were determined by flow cytometry(stained by propidium iodide) and WST-8 respectively. The invasiveness was measured by transwell assay and immunofluorescence technique was used to measure F-actin protein expression. Results Compared with the control group,the mRNA and protein levels of TNFR1 in c6/TNFR1 cell was increased, by 78.5% and 89.7% (P<0.05), respectively.The cell proliferation was inhibited and most of c6/TNFR1 cells were arrested in S+G₂/M phase compared with the control group cells after hyperthermia combined with rhTNF treatment (P<0.05).The fluorescence intensity of F-actin and the average number of C6/TNFR1 cells passing through the inserted filter were decreased by 72.3% and 83.10% respectively, compared to the control group cells after hyperthermia combined with rhTNF treatment (P<0.01).  Conclusion Hyperthermia combined with rhTNF might reduce glioma of C6/TNFR1 invasiveness through blocking cell cycle and reducing the expression of F-action.