Multiple species of Bifidobacterium exhibit the ability to bioconvert conjugated fatty acids (CFAs), which is considered an important pathway for these strains to promote host health. However, there has been limited progress in understanding the enzymatic mechanism of CFA bioconversion by bifidobacteria, despite the increasing number of studies identifying CFA-producing strains. The protein responsible for polyunsaturated fatty acid (PUFA) isomerization in B. breve CCFM683 has recently been discovered and named BBI, providing a starting point for exploring Bifidobacterium isomerases (BIs). This study presents the sequence classification of membrane-bound isomerases from four common Bifidobacterium species that produce CFA. Heterologous expression, purification, and enzymatic studies of the typical sequences revealed that all possess a single c9, t11 isomer as the product and share common features in terms of enzymatic properties and catalytic kinetics. Using molecular docking and alanine scanning, Lys84, Tyr198, Asn202, and Leu245 located in the binding pocket were identified as critical to the catalytic activity, a finding further confirmed by site-directed mutagenesis-based screening assays. Overall, these findings provide insightful knowledge concerning the molecular mechanisms of BIs. This will open up additional opportunities for the use of bifidobacteria and CFAs in probiotic foods and precision nutrition.