Plasmalogens play multiple roles in the structures of biological membranes, cell membrane lipid homeostasis and human diseases. We report the isolation and identification of choline plasmalogens (ChoPlas) from swine liver by high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC)/MS. The growth and viability of hepatoma cells (CBRH7919, HepG2 and SMMC7721) was determined following ChoPlas treatment comparing with that of human normal immortal cell lines (HL7702). Result indicated that ChoPlas inhibited hepatoma cell proliferation with an optimal concentration and time of 25 μmol/L and 24 h. To better understand the mechanism of the ChoPlas-induced inhibition of hepatoma cell proliferation, Caveolin-1 and PI3K/Akt pathway signals, including total Akt, phospho-Akt(pAkt) and Bcl-2 expression in CBRH7919 cells, were determined by western blot. ChoPlas treatment increased Caveolin-1 expression and reduced the expression of phospho-Akt (pAkt) and Bcl-2, downstream targets of the PI3K/Akt pathway. Further cell cycle analysis showed that ChoPlas treatment induced G1 and G1/S phase transition cell cycle arrest. The expression of essential cell cycle regulatory proteins involved in the G1 and G1/S phase transitions, cyclin D, CDK4, cyclin E and CDK2, were also analyzed by western blot. ChoPlas reduced CDK4, cyclin E and CDK2 expression. Taken together, the results indicate that swine liver-derived natural ChoPlas inhibits hepatoma cell proliferation associated with Caveolin-1 and PI3K/Akt signals.