The effect of diet supplementation of Antheraea pernyi (A. pernyi) silk fibroin on the lipid metabolism and antioxidant defense status in high fat-fed mice was investigated. The animals were given normal control diet (NC group), high fat diet (HF group), or high fat diet supplemented with A. pernyi silk fibroin powder (HFS group) for 7 weeks. After the experimental period, the HF group showed significant increase in body weight, plasma and hepatic total cholesterol levels, and hepatic triglyceride concentration, and decreased activities of hepatic antioxidant enzymes relative to NC group. However, the HFS group exhibited marked reduction in body weight, plasma cholesterol and hepatic triglyceride levels, hepatic lipogenic enzyme activities, and lipid peroxidation rate, and higher high-density lipoprotein (HDL)-cholesterol level, fecal triglyceride content, and antioxidant enzymes activities compared with that of HF group. These findings demonstrate that dietary feeding of A. pernyi silk fibroin could improve the lipid metabolism and antioxidant defense system via regulation of hepatic antioxidant and lipogenic enzymes activities. Hence, this silk fibroin may be beneficial as a functional biomaterial for the development of therapeutic agent against high fat diet-induced hyperlipidemia and its related diseases.