ABSTRACT In this study, we identified a P450 enzyme (STH10) and an oxidoreductase (POR) from Thanatephorus cucumeris NBRC 6298 by a combination of transcriptome sequencing and heterologous expression in Pichia pastoris . The biotransformation of 11-deoxycortisol was performed by using Pichia pastoris whole cells coexpressing sth10 and por , and the product analysis indicated that the STH10 enzyme possessed steroidal 19- and 11β-hydroxylase activities. This is a novel fungal P450 enzyme with 19-hydroxylase activity, which is different from the known steroidal aromatase cytochrome P450 19 (CYP19) and CYP11B families of enzymes. IMPORTANCE Hydroxylation is one of the most important reactions in steroid functionalization; in particular, C-19 hydroxylation produces a key intermediate for the synthesis of 19-nor-steroid drugs without a C-19 angular methyl group in three chemoenzymatic steps, in contrast to the current industrial process, which uses 10 chemical reactions. However, hydroxylation of the C-19 angular methyl group remains a very challenging task due to the high level of steric resistance to the C-19 methyl group between the A and B rings. The present report describes a novel fungal P450 enzyme with 19-hydroxylase activity. This opens a new venue for searching effective biocatalysts for the useful process of steroidal C-19 hydroxylation, although further studies for better understanding of the structural basis of the regioselectivity and substrate specificity of this fungal steroidal 19-hydroxylase are warranted to facilitate the engineering of this enzyme for industrial applications.