Separation effect of acetic acid extraction of spleen from Japanese eel, Anguilla japonica by tangential flow ultrafiltration with 3000, 10000 ultons fibre columns, and the two protein samples of the molecular weight (Mw) < 10 ku and the Mw < 3 ku were purified by using the cationic exchange liquid chromatography and together with reverse-phase high-performance liquid chromatography (RP-HPLC). The results showed that three compositions (the protein samples of Mw > 10 ku, Mw <10 ku and Mw <3 ku) could be separated from the acid extraction with two fibre columns. The samples of Mw <10 ku and Mw <3 ku exhibited stronger antibacterial activity than the protein sample of the Mw > 10 ku. The protein sample with the Mw < 10 ku was purified by the cationic exchange liquid chromatography and using pH 9 buffer solution in RP-HPLC, and we obtained an antibacterial peptide with the molecular weight of 1391.82 u when determined by MALDI-TOF MS. The pH 9 and the pH 2 buffer solutions were applied in isolation of the factions obtained from cationic exchange liquid chromatography of the protein sample with the Mw < 3 ku, and we also obtained an antibacterial peptide with the molecular weight of 839.19 u when determined by MALDI-TOF MS. This study suggests that the antibacterial components in the spleen of Anguilla japonica are mostly proteins with low molecular weight.