Fish oil-derived fatty acid ethyl ester (EE) has been widely utilized as a supplement in Europe and United States. However, few studies have assessed the apparent absorption of docosahexaenoic acid (DHA) between non-purified DHA-EE (L-DHAEE) and highly purified DHA-EE (H-DHAEE). This study evaluated the effects of L-DHAEE and H-DHAEE on lipid metabolism and apparent absorption of DHA in rats. Groups of male Wistar rats were fed AIN93G diet containing 2.8% L-DHAEE or 1.0% H-DHAEE for 4 or 8 weeks. As parameters of lipid metabolism and apparent absorption of DHA-EE, serum and liver lipid contents, fecal EE excretion, lipid-metabolizing enzyme activities, and organ fatty acid composition were measured. The L-DHAEE diet increased fecal DHA-EE and total EE excretion compared with the H-DHAEE diet. In rats fed DHA-EE for 4 weeks, H-DHAEE was more efficacious than L-DHAEE at increasing liver DHA content, partly owing to the enhancement of apparent absorption. In addition, rats fed the H-DHAEE diet had higher fatty acid ?-oxidation activity in peroxisomes than rats fed the L-DHAEE diet for 8 weeks. These results suggest that H-DHAEE supplementation may have beneficial functions in the prevention of lifestyle-related diseases as compared with L-DHAEE supplementation.