Poor maternal environments, such as under- or overnutrition, can increase the risk for the development of obesity, type 2 diabetes and cardiovascular disease in offspring. Recent studies in animal models have shown that maternal exercise before and during pregnancy abolishes the age-related development of impaired glucose metabolism, decreased cardiovascular function and increased adiposity; however, the underlying mechanisms for maternal exercise to improve offspring's health have not been identified. In the present study, we identify an exercise-induced increase in the oligosaccharide 3'-sialyllactose (3'-SL) in milk in humans and mice, and show that the beneficial effects of maternal exercise on mouse offspring's metabolic health and cardiac function are mediated by 3'-SL. In global 3'-SL knockout mice (3'-SL), maternal exercise training failed to improve offspring metabolic health or cardiac function in mice. There was no beneficial effect of maternal exercise on wild-type offspring who consumed milk from exercise-trained 3'-SL dams, whereas supplementing 3'-SL during lactation to wild-type mice improved metabolic health and cardiac function in offspring during adulthood. Importantly, supplementation of 3'-SL negated the detrimental effects of a high-fat diet on body composition and metabolism. The present study reveals a critical role for the oligosaccharide 3'-SL in milk to mediate the effects of maternal exercise on offspring's health. 3'-SL supplementation is a potential therapeutic approach to combat the development of obesity, type 2 diabetes and cardiovascular disease.