Microbial a dagger (1) -dehydrogenation is one of the most important transformations in the synthesis of steroid hormones. In this study, a 3-ketosteroid-a dagger(1)-dehydrogenase (kstD(F)) involved in fusidane antibiotic biosynthesis from Aspergillus fumigatus CICC 40167 was characterized for use in steroid transformation. KstD(F) encodes a polypeptide consisting of 637 amino acid residues. It shows 51% amino acid identity with a kstD from Thermomicrobium roseum DSM 5159. Expression of kstD(F) in Escherichia coli and Pichia pastoris showed that all kstD(F) activity is located in the cytoplasm. This indicates that it is a soluble intracytoplasmic enzyme, unlike most kstDs from bacteria, which are membrane-bound. The expression of kstD(F) was performed in P. pastoris, both intracellularly and extracelluarly. The intracellularly expressed protein displayed good activity in steroid transformation, while the extracellularly expressed protein showed nothing. Interestingly, the engineered P. pastoris KM71 (KM71(I)) and GS115 (GS115(I)) showed different transformation activities for 4-androstene-3,17-dione (AD) when kstD(F) was expressed intracellularly. Under the same conditions, KM71(I) was found capable of transforming 1.0 g/l AD to 1,4-androstadiene-3,17-dione (ADD), while GS115(I) could transform 1.5 g/l AD to both ADD and boldenone (BD). The production of BD is attributed to a 17 beta-hydroxysteroid dehydrogenase in P. pastoris GS115(I), which catalyzes the reversible reaction between C17-one and C17-alcohol of steroids. The conversion of AD by GS115(I) and KM71(I) may provide alternative means of preparing ADD or BD. In brief, we show here that kstD(F) is a promising enzyme in steroid a dagger (1) -dehydrogenation that is propitious to construct genetically engineered steroid-transforming recombinants by heterologous overexpression.