[Objective] We screened and identified a strain capable of enantioelectively hydrolyzing rac-α-ethyl-2-oxo-pyrrolidineacetic acid Me ester, followed by using immobilized cells to catalyze the racemic ester. [Methods] We used rac-α-ethyl-2-oxo-pyrrolidineacetic acid Me ester as esterase induction in the medium inoculated with soils to enrich the target microorganism. The strain with high activity for enantioselective hydrolysis of rac-α-ethyl-2-oxo-pyrrolidineacetic acid Me ester was identified by 16S rRNA sequence anal., morphol. observation, physiol. and biochem. properties. We optimized the immobilization conditions of the strain, studied the catalytic properties and operated stability of immobilized cells. [Results] We isolated a gram-neg. bacterial strain which was identified as Methylopila. The optimal immobilization conditions were shown as follows: 0.15% (V/V) of polymine, 0.2% (V/V) of glutaraldehyde, 6 g/L diatomite and 100 g/L cells. Compared with free cells, the optimum pH of immobilized cells changed from 8.0 to 8.5, the optimum temperature changed from 35°C to 40°C, the stability of pH and temperature was improved. Cu2+, Mn2+ and Ca2+ can improve enzyme activity, but Zn2+ and Fe2+ inhibited it. The organic solvent tolerance of immobilized cells was higher than that of free cells. Affinity of immobilized cells with substrates decreased with the increased Km value. We used the immobilized cells to catalyze rac-α-ethyl-2-oxo-pyrrolidineacetic acid Me ester at the concentration of 200 g/L for 20 h, the remaining substrate was (S)-ester with ees > 99.4% and the yield reached 47.8%. The relative activity of the immobilized cells remained 80% after 12 batches. [Conclusion] We established a process to produce (S)-α-ethyl-2-oxo-pyrrolidineacetic acid Me ester from rac-α-ethyl-2-oxo-pyrrolidineacetic acid Me ester by Methylopila sp. cxzy-L013 immobilized cells as the catalyst, which has a great potential for industrialization.