Protamine from salmon spermaries is a novel dietary protein. Chitooligosaccharide (COS) is an oligosaccharide derived from chitin or chitosan, a long-chain polymer, by chemical or enzymatic hydrolysis. These two compounds are known to enhance lipid metabolism by interrupting the digestion and absorption of fat in the body. Cardiovascular disease (CVD) refers to any type of specific disease that affects the heart and circulatory system. Dyslipidemia, a condition involving high levels of low-density lipoprotein (LDL) cholesterol and low levels of high-density lipoprotein (HDL) cholesterol, is generally known to be a primary cause of CVD development. The risk of CVD is usually associated with the atherogenic index (AI) and cardiac risk factor (CRF). The CVD risk is also closely associated with serum levels of total cholesterol (T-CHO), LDL cholesterol and HDL cholesterol. In the present study, we evaluated alterations in serum lipid contents following the administration of protamine, COS and mixtures of these two compounds to male Sprague-Dawley (SD) rats, and their ability to reduce CVD risk. Based on the results of a serum lipid assay, protamine, COS and their mixtures were found to significantly reduce AI, CRF and CVD risk by decreasing serum levels of TG, T-CHO and LDL cholesterol and increasing serum HDL cholesterol levels. By contrast, TG and T-CHO concentrations in feces were markedly increased. Accumulation of lipids in the liver tissues of the SD rats fed high-fat diets was also inhibited by the intake of protamine and COS. Our findings suggest that protamine, COS and combinations of the two compounds may be used as a dietary therapy for preventing CVD due to their suppressive effects on hyperlipidemia and hypercholesterolemia.