<p>Selective utilization of certain cobamides, the vitamin B₁₂-family enzyme cofactors, is a common physiological trait in organohalide respiration, an essential biogeochemical process relevant to global halogen cycling and the bioremediation of chlorinated contaminants. Variations in cobamide structure, particularly the lower base, can disrupt reductive dehalogenase (RDase) catalysis or even completely inactivate organohalide-respiring bacteria (OHRB). Yet, the molecular basis driving such stringent dependence on specific cobamides remains poorly understood. This study demonstrates, using competition-based in vivo assays, that RDase homologs exhibit substantially different cofactor-binding capabilities, directly mirroring the varying cobamide selectivities of their host OHRB. By combining structural modelling with molecular docking, we further identify candidate active-site features and amino acid residues predicted to contribute to cobamide binding and stability within the RDase scaffold. Collectively, our findings link cobamide-utilizing ability in OHRB to RDase architecture and establish an in silico approach for assessing cobamide preference at the enzyme level. Given the extensive diversity of naturally&#xa0;occurring lower bases, computational prediction of cobamide-enzyme compatibility offers a unique opportunity to guide targeted intervention and control of many host-associated and environmental microbiomes (e.g., methanogenic communities).</p>