The multi-step bile acid 7α-dehydroxylating pathway by which a few species of Clostridium convert host primary bile acids to toxic secondary bile acids is of great importance to gut microbiome structure and host physiology and disease. While genes in the oxidative arm of the 7α-dehydroxylating pathway have been identified, genes in the reductive arm of the pathway are still obscure. We identified a candidate flavoprotein-encoding gene predicted to metabolize steroids. This gene was cloned and overexpressed in E. coli and affinity purified. Reaction substrate and product were separated by thin layer chromatography and identified by liquid chromatograph mass spectrometry-ion trap-time of flight (LCMS-IT-TOF). Phylogenetic analysis of the amino acid sequence was performed. We report the identification of a gene encoding a flavoprotein (EDS08212.1) involved in secondary bile acid metabolism by Clostridium scindens ATCC 35704 and related species. Purified rEDS08212.1 catalyzed formation of a product from 3-dehydro-deoxycholic acid that UPLC-IT-TOF-MS analysis suggests loses 4amu. Our phylogeny identified this gene in other bile acid 7α-dehydroxylating bacteria. These data suggest formation of a product, 3-dehydro-4,6-deoxycholic acid, a recognized intermediate in the reductive arm of bile acid 7α-dehydroxylation pathway and the first report of a gene in the reductive arm of the bile acid 7α-dehydroxylating pathway. •A flavoprotein involved in the "reductive arm" of the microbial bile acid 7-dehydroxylation pathway was characterized•Surprisingly, this enzyme catalyzes reduction of two double-bonds•The flavoprotein sequence is conserved among 7α-dehydroxylating bacteria closely related to Clostridium scindens