Fundamental sex-specific differences have been shown to exist in the regulation of metabolism however the underlying mechanisms remain largely unknown. We have recently shown that the resistance to the obesifying effect of a high-fat diet in female C57BL/6J mice is linked both to an ability to significantly reduce their respiratory quotient (RQ), suggesting a greater ability to utilise fat in the diet as a source of fuel, as well as inherent sex differences in the catabolic (POMC/CART) versus anabolic (NPY/AgRP) neurological signalling pathways. One of the main peripheral hormones acting on these neurological signalling pathways to communicate energy status in the body is leptin, an adipocyte-derived hormone primarily secreted in direct proportion to fat mass. By comprehensively investigating the metabolic effect of a physiologically relevant dose of leptin (0.5 mg/kg) in male and female C57BL/6J mice, we now show sex-specific metabolic responses to leptin. Interestingly, the ability of leptin to reduce food intake was only evident in male but not female mice. Energy expenditure was unaltered in both sexes however male but not female mice showed a significant reduction in physical activity. Despite the lack of change observed in female mice in terms of both energy in and energy out, both sexes showed a marked reduction in RQ in response to leptin indicating a greater use of fat as a fuel source. In females only, this reduction in RQ led to a significant reduction in body weight. When leptin was administered at the same time as food was returned following an overnight fast, no significant differences in any parameters were observed in either sex suggesting that fasting over-rides the effects of leptin. Our systematic approach shows that male and female C57BL/6J mice have markedly different metabolic responses to leptin which has important implications for the management of obesity in humans.