The enzyme Δ⁴‐3‐oxosteroid 5β‐reductase (3‐oxo‐5β‐steroid: NADP⁺ oxidoreductase and 4,5β‐dihydrocortisone: NADP⁺Δ⁴‐oxidoreductase) catalyzes the reduction of the Δ⁴ double bond of bile acid intermediates and steroid hormones carrying the Δ⁴‐3‐one structure in the A/B cis configuration. Human Δ⁴‐3‐oxosteroid 5β‐reductase cDNA was isolated from a liver cDNA library by cross hybridization with a previously cloned rat cDNA, which was used as a probe [Onishi, Y., Noshiro, M., Shimosato, T. & Okuda, K.‐I. (1991) FEBS Lett. 283, 215–218]. DNA sequence analysis of a hybridization‐positive clone predicted the human Δ⁴‐3‐oxosteroid 5β‐reductase to contain 326 amino acids. The amino acid sequence of the human Δ⁴‐3‐oxosteroid 5β‐reductase had 79% overall identity to the rat enzyme sequence. It also showed 54% and 50% overall identity with rat 3α‐hydroxysteroid dehydrogenase and human aldose reductase, respectively. RNA blotting analysis demonstrated the existence of a single Δ⁴‐3‐oxosteroid 5β‐reductase mRNA of approximately 2.7 kb in human liver. Transfection of the cDNA into COS cells resulted in the expression of an active enzyme with a high activity toward the bile acid intermediates 7α,12α‐dihydroxy‐4‐cholesten‐3‐one and 7α‐hydroxy‐4‐cholesten‐3‐one. In addition, the expressed enzyme showed a small but significant 5β‐reduction activity toward 11β,17α,21‐trihydroxy‐Δ⁴‐pregnene‐3,20‐dione (cortisol) and 17β‐hydroxy‐Δ⁴‐androsten‐3‐one (testosterone) whereas no activity was observed toward Δ⁴‐pregnene‐3,20‐dione (progesterone) or Δ⁴‐androstene‐3‐17‐dione (androstenedione). The substrate specificity of the human enzyme is considerably narrower than that of the rat enzyme, and the enzyme seems to be more important for bile acid biosynthesis than for metabolism of steroid hormones.