Consumption of excess calories results in obesity and insulin resistance and has been intensively studied in mice and humans. The objective of this study was to determine the specific contribution of dietary fat rather than total caloric intake to the development of obesity associated insulin resistance. We used an intragastric feeding method to overfeed excess calories from low fat diet (and isocalorically matched high fat diet) through a surgically implanted gastric feeding tube to generate obesity in wild-type mice followed by hyperinsulinemic-euglycemic clamp studies to assess the development of insulin resistance. We show that overfeeding low fat diet results in similar levels of obesity as high fat diet feeding in mice. However, despite a similar body weight, obese high fat diet fed mice are more insulin resistant than mice fed an isocaloric low fat diet. Therefore, increased proportion of calories from dietary fat further potentiates insulin resistance in the obese state. Furthermore, crossover diet studies revealed that reduction in dietary fat composition improves glucose tolerance in obesity. In the context of the current obesity and diabetes epidemic it is particularly important to fully understand the role of dietary macronutrients in the potentiation and amelioration of disease.