Aims: To characterize the cause for the lack of conjugated linoleic acid (CLA) reductase (CLA-R) activity in the Butyrivibrio fibrisolvens MDT-5 strain that rapidly isomerizes linoleic acid (LA) to CLA without hydrogenation, the CLA-R was purified and its gene (cla-r) sequence was determined. Methods and Results: CLA-R was purified to near homogeneity as a 53-kDa monomeric protein from the high CLA-R activity-expressing strain MDT-10. The purified CLA-R recognized conjugated double bonds. Unsaturated fatty acids containing 18 carbons markedly increased the CLA-R expression at the transcriptional level. Complete sequencing of the cla-r gene revealed that the CLA-R is a novel protein. Sequence analysis of the cla-r gene from the MDT-5 strain revealed that the MDT-5 CLA-R protein sequence differed from that of the MDT-10 at four consecutive amino acids. Northern and Western blotting analyses confirmed that the cla-r mRNA and protein are expressed normally in MDT-5. Conclusions: Strain MDT-5 expresses the CLA-R protein that lacks enzyme activity because of mutation, which explains why MDT-5 exclusively produces CLA from LA. Significance and Impact of the Study: The cla-r gene was sequenced for the first time. Exogenous fatty acids affected the cla-r transcription. These results will provide additional knowledge on biohydrogenation, and may also augment the CLA production in the gastrointestinal tract.