Abstract Centrifugal partition chromatography (CPC) was applied to separate amphiphilic glycolipids and pseudo-glycolipids synthesized by using cells. Neutral and acidic lipid fractions were isolated by CPC under suitable conditions respectively. Separation of neutral lipid, Gb3-type and Gb4-type oligosaccharide synthesized by using cells, was performed with a two-phase solvent system composed of chloroform–methanol–water at a volume ratio of 5:6:4. On the other hand, separation of acidic lipid, GM3-type oligosaccharide synthesized by using cells, and ganglioside extracted from rat brain were performed with a two-phase solvent system composed of butanol–ethanol–1% acetic acid at a volume ratio of 4:1:5. 8.3 mg of Gb3 analogue, 5.1 mg of Gb4 analogue, and 19.5 mg of GM3 analogue were purified from 3.2 l of culture medium obtained by incubation of African green-monkey kidney (Vero) cells with 50 μM n-dodecyl β-lactoside using CPC.