Skim milk which was hydrolyzed by neutral proteinase was used as medium for Lactobacillus plantarum KLDS1.0391.The effect of different degrees of hydrolysis of skim milk on antibacterial substances production by this strain was studied.Fermentation pH and time was optimized in 3.7 L fermentor by single factor experiment.The peptide distribution of skim milk of different degrees of hydrolysis was analyzed by capillary electrophoresis and SDS-PAGE.Results indicated that casein was completely hydrolyzed and whey was partly hydrolyzed in skim milk with DH of 15%.The peptide of low molecular weight after hydrolyzing was favored by Lactobacillus plantarum KLDS1.0391.Skim milk with DH of 15% was the suitable medium.The optimal constant pH and time for antibacterial substances production were 5.0 and 24 hours.The high level of antibacterial activity achieved 538.53 IU/mL.