17β-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl) is a NADPH dependent member of the short-chain dehydrogenase reductase (SDR) superfamily. Recently, we prepared a homology-built structural model of 17β-HSDcl using the known three-dimensional structure of homologous 1,3,8-trihydroxynaphthalene reductase from the fungus Magnaporthe grisea. This model structure directed our studies of structure–function relationship of the fungal 17β-HSD, as one of the model enzymes of the SDR superfamily. In this work, we investigated the significance of individual amino acid residues for coenzyme and substrate specificity. We performed site directed mutagenesis of R28, a basic residue conserved in most NADPH dependent SDR structures; T200, found only in Streptomyceshydrogenans 3α,20β-HSD and Drosophila alcohol dehydrogenases; and H230, a residue corresponding to the substrate specificity important H221 in human 17β-HSD type 1. All recombinant proteins were expressed in Escherichia coli and purified to homogeneity. Kinetic evaluation of individual mutations was performed by analysis of progress curves of interconversions between 4-estrene-3,17-dione and 4-estrene-17β-ol-3-one, in the presence of NADPH and NADP+; according to the Theorell–Chance reaction mechanism. The results demonstrate the role of the selected amino acid residues; R28 seems to interact with the NADPH 2′-phosphate group; T200 may be involved in binding and dissociation of NADPH/NADP+; while H230 and the neighboring A231 appears not to be responsible for substrate specificity of 17β-HSDcl.