Actinorhodin (ACT) is a benzoisochromanequinone antibiotic produced by Streptomyces coelicolor A3(2), which has served as a favored model organism for comprehensive studies of antibiotic biosynthesis and its regulation. (S)-DNPA undergoes various modifications as an intermediate in the ACT biosynthetic pathway, including enoyl reduction to DDHK. It has been suggested that actVI-ORF2 encodes an enoyl reductase (ER). However, its function has not been characterized in vitro. In this study, biochemical analysis of recombinant ActVI-ORF2 revealed that (S)-DNPA is converted to DDHK in a stereospecific manner with NADPH acting as a cofactor. (R)-DNPA was also reduced to 3-epi-DDHK with the comparable efficacy as (S)-DNPA, suggesting that the stereospecificity of ActVI-ORF2 was not affected by the stereochemistry at the C-3 of DNPA. ActVI-ORF2 is a new example of a discrete ER, which is distantly related to known ERs according to phylogenetic analysis.