Thermal manipulation declined embryonic brain and liver concentrations of leucine (Leu). L-Leu in ovo injection afforded thermotolerance in male broiler chicks. This study aimed to examine the role of in ovo injection of L-Leu in metabolic functions, and differences between male and female broiler chicks in thermotolerance. L-Leu injection was performed in ovo on embryonic day (ED) 7 to reveal its role in metabolic activity in embryos and in post-hatch male and female broiler chicks under heat stress. To examine the metabolic activity of embryos, oxygen (O2) consumption, carbon dioxide (CO2) production, heat production and plasma metabolites were analyzed. Rectal temperature, food intake and plasma metabolites were also analyzed in heat-exposed (35±1 °C for 180 min) male and female broiler chicks. It was found that O2 consumption, heat production, and plasma triacylglycerol (TG) and non-esterified fatty acid (NEFA) concentrations in ED 14 embryos were significantly increased by in ovo injection of L-Leu in comparison with the controls. Plasma glucose concentration was significantly increased in both male and female chicks under heat stress, but in ovo injection of L-Leu attenuated the increase in male chicks. In contrast, plasma TG, NEFA, and ketone body concentrations were significantly higher in male chicks injected in ovo with L-Leu, but not in similarly injected female chicks, compared with control chicks, all under heat stress. Rectal temperature and food intake were significantly lower in male, but not female, chicks under heat stress injected in ovo with L-Leu. In conclusion, in ovo L-Leu administration enhanced the prenatal metabolic rate and lipid metabolisms, which possibly appeared as sex-dependent fashion to facilitate thermotolerance in males. A reduction in heat production through lowered food intake in heat-exposed male, but not female chicks injected in ovo with L-Leu may help to afford thermotolerance in male broiler chicks under heat stress.