Steroidal C19-hydroxylation is pivotal to the synthesis of naturally occurring bioactive C19-OH steroids and 19-norsteroidal pharmaceuticals. However, realizing this transformation is proved to be challenging through either chemical or biological synthesis. Herein, we report a highly efficient method to synthesize 19-OH-cortexolone in 80% efficiency at the multi-gram scale. The obtained C19-OH-cortexolone can be readily transformed to various synthetically useful intermediates including the industrially valuable 19-OH-androstenedione, which can serve as a basis for synthesis of C19-functionalized steroids as well as 19-nor steroidal drugs. Using this biocatalytic C19-hydroxylation method, the unified synthesis of six C19-hydroxylated pregnanes is achieved in just 4 to 9 steps. In addition, the structure of sclerosteroid B is revised on the basis of our synthesis.