(2S,3R)-Dimethyl 1-acetylpiperidine-2,3-dicarboxylate, a valuable intermediate for moxifloxacin, was prepared by CAL B catalyzed kinetic resolution of the racemic diester. The reaction has an optimum temperature of 45–50 °C and an optimum pH of 7.5. It follows typical Michaelis–Menten kinetics with Vmax,obsd = 0.061 ± 0.008 M/h/g, Km,obsd = 0.2 ± 0.045 M at 45 °C. The reaction is highly enantioselective with an enantioselectivity E value of 80 at 45 °C and pH 7.5. Preparative scale enzymatic resolution was carried out in 0.01 M sodium phosphate buffer, pH 7.5 at 45 °C using a substrate loading of 8% (w/w) and a substrate-to-enzyme ratio of 2:1 (w/w). The reaction was complete in 16 h. The enantiomerically pure diester (ee >99%) is obtained in 47.5% yield (95% of theoretical). The unwanted hydrolyzed half ester (ee 87%) was easily converted back to racemic diester in 5 simple steps and 75% overall yield.