The present study was made to determine the optimum pH for isolating antibacterial peptides from the spleen of Japanese eel, Anguilla japonica, by high-performance liquid chromatography (HPLC). The recovery rates of the peptides were investigated by using buffers with three pH values (pH 3, pH 4, and pH 5) in ion-exchange chromatography (IEC) of the spleen protein sample with the molecular weight (MW) < 10 kDa. Following this, for the fractions obtained from IEC, we continued to study the effect of four pH values (pH 2, pH 7, pH 9, and pH 12) on the separation efficiency in reverse-phase high-performance liquid chromatography (RP-HPLC). The results showed using the buffer with pH 4 in IEC could achieve the highest rate of recovery, which was 5.4 fold to that of pH 3 and 1.36 fold to that of pH 5. The treatment at high pH value (pH 9) did significantly affect the chromatographic separation behaviors, which produced more than twice the peak numbers than those in other three pH values. The optimized pH would be helpful for isolating antibacterial peptides from the spleen of eel.