Microbial transformation is an essential component for the commercial production of numerous valuable steroid drugs such as hydrocortisone, prednisolone. Steroid hydroxylation at C11 and C15 positions by filamentous fungi is routinely employed in the steroid industry due to both the necessity of regio- and stereoselective oxyfunctionalization of unactivated C-H bonds of valuable steroid compounds for their desired pharmaceutical activities and also the difficulty of accomplishing such tasks by conventional chemical means. To characterize the steroid hydroxylation system in filamentous fungus Curvularia lunata, which is capable of incorporating a single oxygen atom at C11-βof cortexolone and other derivatives, a NADPH-cytochrome P450 reductase gene was cloned from the fungus based on transcriptome analysis and PCR amplification. The cloned cytochrome P450 reductase gene is predicted to encode a polypeptide of 695 amino acids and exhibits strong similarity to NADPH-cytochrome P450 reductases of several Aspergillus species, with the highest similarity to Aspergillus nidulans observed. The fungal NADPH-cytochrome P450 reductase is an indispensible partner of the microsomal cytochrome P450 hydroxylase by acting as an electron donor. Given the great value of effective 11-βhydroxylation of various steroid compounds for commercial production of valuable steroidal intermediates and drugs, it is anticipated that the cloning and further characterization of the NADPH-cytochrome P450 reductase from C. lunata should facilitate the engineering of industrial microbial strains for efficient drug relevant steroid hydroxylation.