Introduction: Increased phospholipid transfer protein (PLTP) activity has been found to be associated with obesity, and metabolic syndrome in humans.Hypothesis: PLTP might have a direct effect on insulin sensitivity and obesity.Methods: Here we analyzed the effect by using PLTP knockout (PLTP-/-) mouse model.Results: Although, PLTP-/- mice have normal body-weight-gain under chow diet, these mice were protected from high-fat-diet-induced obesity and insulin resistance, compared with wild type mice. In order to understand the mechanism, we evaluated insulin receptor and Akt activation and found that PLTP deficiency significantly enhanced phosphorylated insulin receptor and Akt levels in high-fat-diet fed mouse livers, adipose tissues, and muscles after insulin stimulation, while total Akt and insulin receptor levels were unchanged. Moreover, we found that the deficiency induced significantly more GLUT4 immunostaining in the plasma membranes of adipocytes and muscle cells after insulin stimulation. Finally, we found that PLTP deficient hepatocytes had less sphingomyelins and free cholesterols in the lipid rafts and plasma membranes than that of controls and this may provide a molecular basis for PLTP deficiency-mediated increasing in insulin sensitivity.Conclusions: PLTP deficiency leads to an improvement in tissue and whole-body insulin sensitivity through modulate lipid levels in the plasma membrane, especially in the lipid rafts.