Two members of the human aldo-keto reductase (AKR) superfamily participate in the biosynthesis of bile acids by catalyzing the NADP(H) dependent reduction of 3-keto groups (AKR1C4) and Delta4 double bonds (AKR1D1) of oxysterol precursors. Structure determination of human AKR1C4 and homology modelling of AKR1D1 followed by docking experiments were used to explore active site geometries. Substrate docking resulted in ligand poses satisfying catalytic constraints, and indicates a critical role for Trp227/230 in positioning the substrate in a catalytically competent orientation. Based on the evidence gathered from our docking experiments and experimental structures, this tryptophan residue emerges as a major determinant governing substrate specificity of a subset of enzymes belonging to the AKR1 subfamily.