A steroid 5β-reductase gene corresponding to the hypothetical protein of LOC100247199 from leaves of Vitis vinifera (var. ‘Chardonnay’) was cloned and overexpressed in E. coli. The recombinant protein showed 5β-reductase activity when progesterone was used as a substrate. The reaction was stereo-selective, producing only 5β-products such as 5β-pregnane-3,20-dione. Other small substrates (terpenoids and enones) were also accepted as substrates, indicating the highly promiscuous character of the enzyme class. Our results show that the steroid 5β-reductase gene, encoding an orthologous enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in leaves of the cardenolide-free plant Vitis vinifera. We emphasize the fact that, on some occasions, different reductases (e.g., progesterone 5β-reductase and monoterpenoid reductase) can also use molecules that are similar to the final products as a substrate. Therefore, in planta, the different reductases may contribute to the immense number of diverse small natural products finally leading to the flavor of wine. KEYWORDS: Vitis vinifera, enone 5β-reductase, secondary metabolites, biosynthesis, gene evolution