Methylmalonyl-CoA mutase is an adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to the cobalt in solution, is replaced by H610 donated by the active site. The contribution of the lower axial base to the ca. trillion-fold rate acceleration of the homolytic cleavage of the upper axial cobalt-carbon bond has been the subject of intense scrutiny in the model inorganic literature. In contrast, trans ligand effects in methylmalonyl-CoA mutase and indeed, the significance of the ligand replacement are poorly understood. In this study, we have used site directed mutagenesis to create the H610A and H610N variants of methylmalonyl-CoA mutase and report that both mutations exhibit both diminished activity (5000- and 40,000-fold respectively) and profoundly weakened affinity for the native cofactor, AdoCbl. In contrast, binding of the truncated cofactor analog, adenosylcobinamide, lacking the nucleotide tail, is less impaired. The catalytic failure of the H610 mutants is in marked contrast to the phenotype of the adenosylcobinamide-GDP reconstituted wild type enzyme that exhibits only a four-fold decrease in activity although H610 fails to coordinate when this cofactor analog is bound. Together, these studies suggest that H610 may (i) play a structural role in organizing a high affinity cofactor binding site possibly via electrostatic interactions with D608 and K604, as suggested by the crystal structure and (ii) play a role in catalyzing the displacement of dimethylbenzimidazole thereby facilitating the conformational change that must precede cofactor docking to the mutase active site.