Objective To explore whether heat acclimation can protect the structure and function of mitochondrion of cerebral cortex and myocardium in rats with moderate and high intensity exercise in hygrothermal environment, and to explore its protective mechanism. Methods Forty SD rats underwent swimming experiment bearing weight and 8 rats were eliminated due to too short-swim or long-swim time. The rest 32 rats were randomly divided into four groups (n=8): room-temperature activity (RA) group, room-temperature exercise (RE) group, high-temperature activity (HA) group and high-temperature exercise (HE) group. Rats in RA and HA groups were placed at room temperature ([24±1]℃) and high temperature ([32±1]℃) to have a free activity for 2 h/d. Rats in RE and HE groups were placed at room temperature ([24±1]℃) and high temperature ([32±1]℃) to have a free activity for 1 h/d and then were allowed to run at a constant speed on the animal treadmill for 1 h/d (slope: 0°, speed: 14 m/min). After 14 days the heat acclimation rat models were created. Rats in all groups underwent moderate and high intensity exercise in hygrothermal environment for 30 min on the 3^rd day. The body weight loss rate and rectal temperatures of rats were measured before and after high and medium intensity exercise in hygrothermal environment. The contents of plasma norepinephrine (NE), arginine vasopressin (AVP) and cortisol (Cort) in rats were detected by radioimmunoassay. The ultrastructure of the mitochondria of cerebral cortex and myocardium was observed by transmission electron microscopy. The contents of ATP synthase and reactive oxygen species (ROS) were detected by ELISA method and the total antioxidative capacity was measured by ABTS method. Results The rectal temperatures before and after stimulation and the ascending range of rectal temperatures of rats in HE group were significantly lower than those in the RA, RE and HA groups (P<0.05, P<0.01). After stimulation with moderate and high intensity exercise in hygrothermal environment, the body weight loss rate of rats in HE group was significantly higher than that in RA group (P<0.01), the contents of plasma Cort and ROS of rats in HE group were significantly lower than those in the RA, RE and HA groups (P<0.05, P<0.01), the content of ATP synthase in mitochondrion and total antioxidative capacity in tissue from cerebral cortex and myocardium of rats in HE group were significantly higher than those in the RA, RE and HA groups (P<0.01), and the mitochondrion and synaptic structures in the cerebral cortex and the structures of myocardium, Z line and transverse tubule in the myocardium in HE group were superior to those in the RA, RE and HA groups. Conclusion Heat acclimation can enchance the oxidative and respiratory function of mitochondria and reduce the level of cell oxidative stress and the content of plasma Cort, so as to protect the mitochondrial structure of cerebral cortical neurons and cardiomyocytes in rats with moderate and high intensity exercise in hygrothermal environment.