Protamine has been widely used as a pharmaceutical product and natural food preservative. However, few studies have been conducted to assess the beneficial function of dietary protamine. This study examined the effects of dietary salmon protamine on serum and liver lipid levels and the expression levels of genes encoding proteins involved in lipid homeostasis in the liver of rats. Groups of male Wistar rats were fed AIN93G diet containing 2% or 5% protamine. After 4 weeks of feeding these diets, markedly decreased serum and liver cholesterol (CHOL) and triacylglycerol levels were noted. Increased activity of liver carnitine palmitoyltransferase-2 and acyl-CoA oxidase, which are key enzymes of fatty acid ${eta}$-oxidation in the mitochondria and peroxisomes, was found in rats fed on protamine. Furthermore, rats fed protamine showed enhanced fecal excretion of CHOL and bile acid and increased liver mRNA expression levels of ATP-binding cassette (ABC) G5 and ABCG8, which form heterodimers and play a major role in the secretion of CHOL into bile. The decrease in triacylglycerol levels in protamine-fed rats was due to the enhancement of liver ${eta}$-oxidation. Furthermore, rats fed protamine exhibited decreased CHOL levels through the suppression of CHOL and bile acid absorption and the enhancement of CHOL secretion into bile. These results suggest that dietary protamine has beneficial effects that may aid in the prevention of lifestyle-related diseases such as hyperlipidemia and atherosclerosis.