Glucose is a principle precursor for milk lactose and de novo synthesis of milk fat; therefore exercising during lactation could create competition for glucose between exercising muscle and lactating mammary gland. This study investigated the combined effects of maternal dietary glucose (20%, 40%, 60%), exercise (chronically exercised, sedentary) and litter size (8, 12 pups) on rat mammary gland composition, milk composition, milk yield and pup growth. Chronic exercise increased milk fat concentrations and an interaction between chronic exercise and 20% dietary glucose decreased milk lactose concentrations compared to 40% or 60% glucose diets. Restricting maternal dietary glucose also decreased milk fat concentrations and exercise decreased mammary fat. In addition, pups of dams fed the 40% glucose diet were heavier on lactation day 15 than pups of dams fed the 60% diet. These results suggest that competition for glucose occurs and that a 40% glucose maternal diet may be more appropriate for pup growth.