As a kind of insect oil, silkworm (Bombyx mori) pupal oil (SPO) is rich in abundant nutritive components including unsaturated fatty acids especially alpha-linolenic acid, and can bring significant economic benefits. SPO extracted by supercritical carbon dioxide from desilked silkworm pupae was employed to investigate the beneficial effects of SPO on growth parameters and lipid metabolism of rats fed with high-fat diet (HFD). Results demonstrated that SPO supplementation could reduce the body weight gain and relative liver weight and it could lower the total cholesterol and triglycerides in serum and liver in HFD fed rats. It significantly decreased serum low-density lipoprotein-cholesterol (LDL-C) levels and increased serum and hepatic high-density lipoprotein-cholesterol (HDL-C) level in HFD rats. Hepatic protection by SPO was confirmed by histological analysis of the liver via H&E staining. SPO relieved the negative effect of HFD on the mRNA expression of lipoprotein lipase (LPL), toll-like receptor 4, and nuclear factor-kappa B p65 in the liver. Our study indicates that SPO is a promising dietary supplement regulating lipid metabolism and potentially contributes to the prevention and therapy of fatty liver diseases such as obesity-related atherosclerosis.