Despite the progress in use of hyperthermia in clinical practice, the thermosensitivity of cancer cells is poorly understood. In a previous study, we found that sensitivity to hyperthermia varied among ovarian cancer cell lines. Upon hyperthermia, glycolytic enzymes decreased in hyperthermia-resistant SKOV3 cells. However, the mechanisms of glycolysis inhibition and their relationship with thermoresistance remain to be explored. In this study, metabolomic analysis indicated downregulation of glycolytic metabolites in SKOV3 cells after hyperthermia. Proteomic and pathway analyses predicted that the ubiquitin pathway was specifically activated in resistant SKOV3 cells, compared with hyperthermia-sensitive A2780 cells, and STUB1, a ubiquitin ligase, potentially targeted PKM, a glycolytic rate-limiting enzyme. PKM was found to be degraded via ubiquitination upon hyperthermia. Although glycolysis was inactivated by hyperthermia, ATP production was maintained. We observed that oxygen consumption and mitochondrial membrane potential were activated in SKOV3 cells but suppressed in A2780 cells. The activation of mitochondria could compensate for the ATP production lost due to suppression of glycolysis by hyperthermia. Although the physiological significance has not yet been elucidated, our results demonstrated that metabolomic adaptation from the Warburg effect to mitochondrial oxidative phosphorylation could contribute to thermoresistance in ovarian cancer cells.