Background and aims: Liver toxicity is a well-documented and potentially fatal adverse complication of hyperthermia. However, the impact of hyperthermia on the hepatic metabolome has hitherto not been investigated. Methods: In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was applied to assess the in vitro metabolic response of primary mouse hepatocytes (PMH, n = 10) to a heat stress stimulus, i.e., after 24 h exposure to 40.5 °C. Metabolomic profiling of both intracellular metabolites and volatile metabolites in the extracellular medium of PMH was performed. Results: Multivariate analysis showed alterations in levels of 22 intra- and 59 extracellular metabolites, unveiling the capability of the metabolic pattern to discriminate cells exposed to heat stress from cells incubated at normothermic conditions (37 °C). Hyperthermia caused a considerable loss of cell viability that was accompanied by significant alterations in the tricarboxylic acid cycle, amino acids metabolism, urea cycle, glutamate metabolism, pentose phosphate pathway, and in the volatile signature associated with the lipid peroxidation process. Conclusion: These results provide novel insights into the mechanisms underlying hyperthermia-induced hepatocellular damage.