An aldo-keto reductase gene (klakr) from Kluyveromyces lactis XP1461 was cloned and heterologously expressed in Escherichia coli. The aldo-keto reductase KlAKR was purified and found to be NADH-dependent with a molecular weight of approximately 36kDa. It is active and stable at 30°C and pH 7.0. The maximal reaction rate (vmax), apparent Michaelis–Menten constant (Km) for NADH and t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a) and catalytic number (kcat) were calculated as 7.63Umg−1, 0.204mM, 4.42mM and 697.4min−1, respectively. Moreover, the KlAKR has broad substrate specificity to a range of aldehydes, ketones and keto-esters, producing chiral alcohol with e.e. or d.e. >99% for the majority of test substrates.