A carbonyl reductase gene (yueD) from Bacillus sp. ECU0013 was heterologously overexpressed in Escherichia coli, and the encoded protein (BYueD) was purified to homogeneity and characterized. The NADPH-dependent reductase showed a broad substrate spectrum towards different aromatic ketones, and α- and β-ketoesters. Although the enantioselectivity was high to moderate for the reduction of α-ketoesters, all the tested β-ketoesters and aromatic ketones were reduced to the corresponding chiral alcohols in enantiomerically pure forms. Furthermore, the practical applicability of this enzyme was evaluated for the reduction of ethyl 4-chloro-3-oxobutanoate (1a). Using Escherichia coli cells coexpressing BYueD and glucose dehydrogenase, 215 g L(-1) (1.3 M) of 1a was stoichiometrically converted to ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-1b) in an aqueous-toluene biphasic system by using a substrate fed-batch strategy, resulting in an overall hydroxyl product yield of 91.7% with enantiomeric purity of 99.6% ee.