OBJECTIVE : This study reports the identification of a new bacterial azoreductase (AzoR) from Klebsiella oxytoca GS-4-08, its heterologous production in Escherichia coli and the decolorization of azo dyes. RESULTS : The AzoR, as a flavin-free, oxygen-insensitive enzyme, has a molecular mass of 22 kDa and a high substrate specificity for methyl red (MR). In the presence of 5 mM of NADH, the enzyme activity for decolorization of 50 mg l-1 of MR reached 5.61 μmol min-1 mg protein-1. Double-reciprocal plots indicated that the NADH and MR reductions proceed by ping-pong mechanism. The calculated Vm was 8.17 μM min-1 mg protein-1, and the AzoR retained over 75% of relative activity under temperature range from 20 to 50 °C. CONCLUSIONS : This study for the first time identified an AzoR from Klebsiella oxytoca strain, which enabled efficient degradation of azo dyes by strain GS-4-08.