Sertoli cells (SCs) provide structural and nutritional support for developing germ cells. Normal glucose metabolism of SCs is necessary for spermatogenesis. Melatonin could alleviate the effects of heat stress on spermatogenesis. However, the influences of heat stress on glucose metabolism in SCs remain unclear, and the potential protective mechanisms of melatonin on SCs need more exploration. In this study, boar SCs were treated at 43°C for 30 min, and different concentrations of melatonin were added to protect SCs from heat stress‐induced impairment. These results showed that heat stress‐induced oxidative stress caused cell apoptosis, inhibited the pentose phosphate pathway, and decreased the ATP content. Furthermore, heat stress increased the expressions of glucose intake‐ and glycolytic‐related enzymes, which enhanced the glycolysis activity to compensate for the energy deficit. Melatonin relieved heat stress‐induced oxidative stress and apoptosis by activating the Kelch‐like ECH‐associated protein 1 (KEAP1)/NF‐E2‐related factor 2 signaling pathway to increase the capacity of antioxidants. In addition, melatonin enhanced heat‐shock protein 90 (HSP90) expression through melatonin receptor 1B (MTNR1B), thereby stabilizing hypoxia‐inducible factor‐1α (HIF‐1α). Activation of the HIF‐1α signaling pathway enhanced glycolysis, promoted the pentose phosphate pathway, and increased cell viability. Our results suggest that melatonin reprograms glucose metabolism in SCs through the MTNR1B–HSP90–HIF‐1α axis and provides a theoretical basis for preventing heat stress injury.