L-Xylulose reductase (XR) catalyzes the oxidoreduction between xylitol and L-xylulose in the uronate cycle. The enzyme has been shown to be identical to diacetyl reductase, an enzyme that reduces alpha-dicarbonyl compounds. XR belongs to the short-chain dehydrogenase/reductase family, and shows high sequence identity with mouse lung carbonyl reductase (MLCR), an enzyme that reduces 3-ketosteroids but not sugars. In this study, we have confirmed the roles of Ser136, Tyr149 and Lys153 of XR as the catalytic triad by drastic loss of activity resulting from the mutagenesis of S136A, Y149F and K153M in rat XR. We have also constructed several mutant XRs, in which putative substrate binding residues from rat XR were substituted with those found in the corresponding positions of MLCR, in order to identify amino acids responsible for the different substrate recognition of the enzymes. While single mutants at positions 137, 143, 146, 190 and 191 caused little or moderate change in substrate specificity, a double mutant (N190V and W191S) and triple mutant (Q137M, L143F and H146L) resulted in almost loss of activity for only the sugars. In addition, the triple mutant exhibited 3-ketosteroid reductase activity, which was further enhanced by quintuple mutagenesis of the above five residues. These results suggest the importance of the size and hydrophobicity of the five residues for substrate recognition by XR and MLCR. Furthermore, the mutant enzymes containing a Q137M mutation were stable against cooling, which provides a structural mechanism of the cold inactivation that is a characteristic of the rodent XR.