Gliomas are a group of heterogeneous primary central nervous system tumors. Hyperthermia has provento be a potential therapeutic tool for cancers in the clinic. However, the molecular mechanisms of hyperthermiaremain unclear. The objective of this study was to investigate the effects of hyperthermia on the invasivenessin C6 glioma cells and related molecular pathways. Here our data show hyperthermia stimulated the release oftumor necrosis factor-alpha (TNF-α) and decreased C6 glioma cell migration and invasive capability at 30, 60,120 and 180 min; with increased spontaneous apoptosis in C6 glioma cells at 120 min. We also found mitogenactivatedprotein kinase (P38 MAPK) protein expression to be increased and nuclear factor-kappa B (NF-κB)protein expression decreased. Based on the results, we conclude that hyperthermia alone reduced invasion ofC6 glioma cells through stimulating TNF-α signaling to activate apoptosis, enhancing P38 MAPK expressionand inhibiting the NF-κB pathway, a first report in C6 rat glioma cells.