SUMMARYCaloric restriction (CR) is a nutritional intervention that reduces the risk of age-related diseases in numerous species, including humans. CR’s metabolic effects, including decreased fat mass and improved insulin sensitivity, play an important role in its broader health benefits. However, the extent and basis of sex differences in CR’s health benefits are unknown. We found that 30% CR in young (3-month-old) male mice decreased fat mass and improved glucose tolerance and insulin sensitivity, whereas these effects were blunted or absent in young female mice. Females’ resistance to fat and weight loss was associated with decreased lipolysis, lower systemic energy expenditure and fatty acid oxidation, and increased postprandial lipogenesis compared to males. Positron emission tomography-computed tomography (PET/CT) with18F-fluorodeoxyglucose (18F-FDG) showed that peripheral glucose uptake was comparable between sexes. Instead, the sex differences in glucose homeostasis were associated with altered hepatic ceramide content and substrate metabolism: compared to CR males, CR females had lower TCA cycle activity but higher blood ketone concentrations, a marker of hepatic acetyl-CoA content. This suggests that males use hepatic acetyl-CoA for the TCA cycle whereas in females it accumulates, thereby stimulating gluconeogenesis and limiting hypoglycaemia during CR. In aged mice (18-months old), when females are anoestrus, CR decreased fat mass and improved glucose homeostasis to a similar extent in both sexes. Finally, in a cohort of overweight and obese humans CR-induced fat loss was also sex- and age-dependent: younger females (<45 years) resisted fat loss compared to younger males while in older subjects (>45 years) this sex difference was absent. Collectively, these studies identify age-dependent sex differences in the metabolic effects of CR and highlight adipose tissue, the liver and oestrogen as key determinants of CR’s metabolic benefits. These findings have important implications for understanding the interplay between diet and health and for maximising the benefits of CR in humans.HIGHLIGHTSCaloric restriction (CR) decreases fat mass and improves glucose homeostasis in young male mice, but young females resist these effects.CR females resist lipolysis, decrease energy expenditure and increase postprandial lipogenesis more than CR males, explaining how females resist fat loss.Sex differences in glucose homeostasis are associated with altered hepatic metabolism and gluconeogenesis, without marked differences in peripheral glucose uptake.CR’s effects on fat loss and glucose homeostasis are comparable in aged male and female mice, implicating oestrogen as the driver of the sexually dimorphic effects in young mice.In humans, females resist CR-induced fat loss in an age-dependent manner, further supporting the role of oestrogen in the sexually dimorphic effects of CR.