Background: Heat stress is one of the most important environmental stressors challenging poultry production worldwide. Understanding and controlling environmental stressors is crucial for successful poultry production. OBJECTIVES: The aim of the present study was to investigate the effects of melatonin supplementation and darkness regime on reducing the negative effects of heat stress in broiler chickens. METHODS: A total of 400 broiler chickens (Arian) were obtained and subjected to four different groups. The control chickens were grown in normal conditions without receiving any treatment. The positive control birds were grown similar to the control but exposed to an acute heat stress for 6 h/d from days 35 to 40. The dark group chickens were reared under a dark schedule and exposed to heat stress. The birds of the fourth group were reared similar to the positive control but received 40 ppm melatonin in the diet from days 30 to 40 of age. Dead birds were autopsied. The weekly growth performance of chickens was determined and blood samples were taken for hormonal analysis. RESULTS: The number of dead birds due to heat stress was significantly decreased in chickens subjected to the dark program and/or those which received melatonin in the diet compared with those of positive control chickens. Such differences were accompanied with the significantly lower levels of plasma T3, T3/T4 and corticosterone in these groups of chickens. Additionally, plasma thiobarbituric acid reacting substances level was significantly higher in positive control broilers compared to their counterparts. CONCLUSIONS: Our findings indicate a reduction of metabolic rate and heat production in dark regime and melatonin supplementation that can reduce the side effects of heat stress and, therefore, reduce the mortality rate.