The purpose of this study was to investigate the influence of four kinds of Lactic acid bacteria (LAB) on stereoselective degradation of theta-cypermethrin (CYP), including Lactobacillus plantarum, Lactobacillus casei, Lactobacillus delbrueckii, and Streptococcus thermophilus. An effective analytical method for (±)-theta-CYP in medium was developed by high-performance liquid chromatography with cellulose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase. theta-Cypermethrin was spiked to LAB medium with different inoculation rates and sampled at 0, 2, 8, 24, 36, 48, 72, 120, 168, and 240 hours. The results showed that LAB influenced the half-lives and enantiomer fractions of theta-CYP enantiomers, which lead a closer degradation rate between the 2 stereoisomers, and no obvious difference was found among 4 LABs. Besides, the stereoselective degradation of theta-CYP was closely related to pH. The lower the pH (pH of 3, 5, 7, and 9), the lower the enantiomer fraction (from 4.88 to 6.69). At pH of 3, 7, and 9, significant differences of half-lives between enantiomers were observed. (-)-theta-Cypermethrin decreased faster than (+)-theta-CYP under pH of 3, while opposite results were indicated under pH of 7 and 9. Moreover, the acidic condition contributed to the higher chiral configuration stability of (±)-theta-CYP. (+)-Enantiomer was influenced by pH in a greater degree than (-)-enantiomer.