This study aimed to investigate the thermal responses of acclimated workers exposed to heat stress in a real work environment. The physiological indices and subjective thermal perception of the 14 acclimated workers were measured in an industrial plant. The effects of wet bulb globe temperature (WBGT) on physiological indices and subjective thermal perception were studied. The differences in thermal responses between the acclimated workers and unacclimated college students exposed to heat stress were compared and analyzed. The relationship between the mean skin temperature and the thermal sensation was revealed. The results show that the mean skin temperature, oral temperature, and heart rate of the acclimated workers increase with WBGT, while the blood pressure decreases with WBGT. Compared with the unacclimated college students, the acclimated workers felt more comfortable and tolerant under the same heat stress. The thermal neutral mean skin temperature of the acclimated workers is 32.3 °C, which is approximately 1.0 °C lower than that of the unacclimated college students. The results of this study can help ensure the occupational safety and health of heat stress-exposed workers.