3-ketosteroid-9alpha-hydroxylase (KSH), a key enzyme in the microbial steroid degradation, is highly valuable for the production of some steroid drugs. Degenerate primers were designed by comparing the ksh from Rhodococcus erythropolis SQ1 and its homologous sequences in the reported genome of Mycobacteria. Subsequently, a gene fragment of KSH was cloned from Mycobacterium sp. NwIB-01, a sterol-transforming bacterium isolated from soil in our lab. According to the conservative sequence, the full-length 1188 bp gene encoding ksh (designated as M.S.-ksh) was obtained by chromosome walking, which showed 85% identity with the ksh of M. smegmatis mc(2)155. The heterologous expression of KSH was achieved in Escherichia coli BL21(DE3) using the pET-32a-c(+) vector system. The expressed KSH protein was mostly in soluble form after IPTG induction at 30 degreesC and accounted for more than 30% of total bacterial proteins according to SDS-PAGE electrophoresis. The molecular mass of KSH was about 45 kD, which was exactly the size predicted. After Ni2+ affinity chromatography, the purity of the target protein was more than 90%. Our work will definitely contribute to the industrial production of some steroid drugs by developing KSH genetically engineered bacteria.