A new method for the separation and determination of L- and D-lactic acid in pickles supernatants by high-performance liquid chromatography (HPLC) with a chiral colomn was studied here. The culture supernatants of pickles were centrifuged for 20 min at 8000 r/min and the supernatant fractions were filtered through a 0.45 µm membrane. The samples were then analyzed by HPLC on chiral columns with a PDA detector at 254 nm. High separation efficiency was achieved by using a Chirex 3126 (D)-penicillami column (4.6 mm ID×250 mm L, 5 µm) and a mobile phase consisting of CuSO4 (2 mmol/L) and isopropyl alcohol-water (5:95). The calibration curves of L-and R- lactic acid were linear with regressions being of 0.999 in the concentration range of 2.5~20.0 g/L. The average recovery was 99.82% for D--lactic acid and 100.02% for L-lactic acid in filtrate of pickles. The method was simple and easy to operate with high accuracy and reproducibility and it was suitable for detection the concentration of L- and D-lactic acid in pickles.