High-speed countercurrent chromatography (HSCCC) was applied to purification of fish oil ethyl esters (FOEE). The CCC separations were performed using non-aqueous two-phase solvent systems in two steps: The first purification was carried out with a hexane-acetonitrile system where 30 mL of crude FOEE was cleaned in 140 min yielding 23.1 mL of refined FOEE. The product was colorless and free of saturated fatty acid esters. The second and final purification was performed with a hexane-dichloromethane-acetonitrile (5 : 1:4, v/v/v) where 1 g of the refined FOEE was resolved into three fractions, ethyl hexadecenoate, ethyl octadecenoate and the third composed of 56.4% of ethyl docosahexaenoate and 39.3% of ethyl eicosapentenoate. The separation was completed in 145 min. However, polyunsaturated fatty acids are not resolved with this solvent system due to their low partition coefficients.