Dietary fat is considered an important contributing factor in the obesity epidemic, and high-fat diets are used widely to induce obesity and diabetes-related traits in susceptible rodent strains. Little attention, however, is usually paid to the interaction of fat with the other macronutrients. The aim of this study, therefore, was to investigate the effects of high-fat, isoenergetic diets with different protein:carbohydrate (CHO) ratios on obesity, energy metabolism, and glucose homeostasis in mice. Male adult C57BL/6J mice consumed ad libitum for 10 wk a control diet (41:42:17 ratio of CHO:protein:fat, 15.5 kJ/g) or 2 different high-fat diets: high carbohydrate (HC; 41:16:43, 17.7 kJ/g) or low carbohydrate (LC; 11:45:44, 17.5 kJ/g). Body weight and fat gains were rapid and were greater in HC mice than in other groups due to an initial pronounced hyperphagia and subsequent passive overconsumption. Weight and fat gains were less in LC mice but still greater than in controls. Energy expenditure was not affected by the diets, and total energy intake explained 84% of the variation in final body weight. The respiratory quotient was lower in LC mice than in other groups, indicating high fat oxidation rates due to the LC diet. Blood glucose was lower and insulin sensitivity greater in LC mice than in HC mice. We conclude that increasing the protein:CHO ratio in a high-fat diet delays but does not prevent the development of adiposity. However, glucose homeostasis was improved in LC mice, indicating that a combination of high fat and high CHO is responsible for the development of metabolic syndrome-related traits in mice.