Objective To investigate the protective effect of heat acclimation on hyperthermia-induced liver injury in mice and the associated changes in the kynurenine pathway in the liver. Methods (1) The heat acclimation (HA) model was constructed. Thirty-six male C57BL/6J mice were randomly divided into four groups control group(Ctrl, not subjected to heatstroke), HA group ( subjected to heat acclimation but not to heatstroke), Ctrl +heat stress (HS )group ( only subjected to HS ) and HA + HS group ( subjected to HA and HS ). Core body temperature was monitored once a week. Blood and liver specimens were collected after four weeks. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured. The pathological changes in the liver and survival time under hyperthermia conditions were observated. Untargeted metabolomics was used to analyze hepatic metabolic changes. Kynurenic acid (KYNA) and other metabolites in the hepatic kynurenine pathway (KP) were measured. Real-time quantitative PCR (Q-PCR) was used to detect the mRNA expressions of the key enzymes in the hepatic KP. Protein immunoblotting was used to detect the corresponding protein levels. (2) The HS model after Kyat2 inhibition was constructed. Thirty-six male C57BL/6J micewere randomly divided into four groups: Ctrl group, Ctrl+HS group, Kyat2 inhibitor PF-04859989 intervention (PF) group, PF+ HS group. The PF and PF+HS groups were injected intraperitoneally with PF-04859989 10mg/kg, and the remaining two groups were injected intraperitoneally with an equal volume of DMSO solution. HS or sampling was initiated 1 h after intervention. Blood and liver specimens were collected from mice 6 h after recovery from HS, and the activities of ALT, AST and lactate dehydrogenase (LDH) were detected. The survival time under hyperthermia conditions was observated. The expression level of Kyat2 in the liver was detected by Q-PCR. The corresponding protein level was detected by protein immunoblotting. Results (1) Compared with the Ctrl+HS group, serum ALT and AST levels of the HA+HS group were significantly lower, and the survival time was significantly prolonged. Compared with the Ctrl group, the levels of KYNA in the serum and KP metabolites 3-hydroxy-phthalamidobenzoic acid （3-HAA） and pyridinecarboxylic acid（PA） in the liver were significantly higher in the HA group, whereas the levels of KYNA, L-Trp, and L-kyn in the liver were significantly lower. The expressions of Afmid、Kynu and Kmo were higher than Ctrl, whereas the expression of Kyat2 enzyme in the liver was significantly lower. (2) Compared with the Ctrl group, there were no significant differences in serum ALT, AST, LDH and survival time in the PF group. Compared with the Ctrl+HS group, the PF+HS group displayed significantly prolonged survival time, and serum ALT, AST, and LDH were significantly reduced. Conclusion Tryptophan metabolism through KP was enhanced in the liver of heat-acclimated mice. Inhibition of kynurenic acid production attenuated hyperthermia-induced liver injury in mice.