High-fat diet (HFD) induces obesity. This study examined the effects of Shiitake mushroom on the prevention of alterations of plasma lipid profiles, fat deposition, energy efficiency, and body fat index induced by HFD. Rats were given a low, medium, and high (7, 20, 60 g/kg = LD-M, MD-M, HD-M) Shiitake mushroom powder in their high-fat (50% in kcal) diets for 6 weeks. The results showed that the rats on the HD-M diet had the lowest body weight gain compared to MD-M and LD-M groups (P < 0.05). The total fat deposition was significantly lower (-35%, P < 0.05) in rats fed an HD-M diet than that of HFD group. Interestingly, plasma triacylglycerol (TAG) level was significantly lower (-55%, P < 0.05) in rats on HD-M than HFD. This study also revealed the existence of negative correlations between the amount of Shiitake mushroom supplementation and body weight gain, plasma TAG, and total fat masses.