Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15 alpha-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15 alpha-hydroxylation, the gene encoding the steroid 15 alpha-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15 alpha-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15 alpha-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15 alpha-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15 alpha-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15 alpha-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15 alpha-hydroxylation reactions.