Background: Heatwave events occur more frequently, accompanied by a significant increase in the ambient concentration of fine particulate matter (PM2.5). Epidemiological and experimental studies have suggested that heat stress or PM2.5 exposure would impair glucose homeostasis and insulin sensitivity. Objective: To explore the joint effects of heat stress and PM2.5 exposure on glucose metabolism and hepatic insulin pathway. Methods: C57BL/6 mice were randomly divided into filtered air (FA) group, fine particulate matter (PM) group, filtered air combined with heat stress (FH) group, and PM combined with heat stress (PH) group. Mice were exposed to PM for a total of 6 weeks, and heat stress combined with PM exposure was applied at the 5th and 6th weeks via a whole-body exposure system. Systemic glucose homeostasis, insulin sensitivity, and circulating inflammatory cytokines were examined. HSP72 expression and insulin signaling in the liver were measured. Results: Glucose tolerance and insulin sensitivity were impaired in response to heat stress, accompanied by lessened hepatic GLUT2 expression and inhibited insulin signaling pathway. No synergistic effects of heat stress and PM2.5 exposure on glucose homeostasis were observed, while heat-upregulated HSP72 expression was attenuated with accumulated TNFα induced by further PM2.5 exposure. Conclusion: Heat stress combined with PM2.5 exposure induced TNFα, which could inhibit heat-elevated hepatic HSP72 expression. Elevated circulating TNFα impaired hepatic insulin signaling and GLUT2 expression. Then, glucose homeostasis was perturbed, and insulin action was impaired.