An increasing number of studies indicate that each step of the intracellular processing of vitamin B12 or cobalamin (Cbl) involves protein-protein interactions. We have previously described a novel interaction between methionine synthase (MS) and MMACHC and its effect on the regulation of MMACHC activity. Our goal is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. We dissected the interactions and their alterations by co-immunoprecipitation and DuoLink proximity ligation assays in fibroblasts with cblG, cblE, and cblC genetic defects affecting respectively the expression of MS, methionine synthase reductase (MSR) and MMACHC and in HepG2 cells transfected with corresponding siRNAs. We observed the known interactions of MS with MSR and with MMACHC as well as MMADHC with MMACHC, but we also observed novel interactions for MSR with MMACHC and with MMADHC and MS with MMADHC. Furthermore, we show that the absence of MS or MMACHC expression disrupts the interactions between the other interactome members, in cblC and cblG fibroblasts and in HepG2 cells transfected with siRNAs. Our data show that the processing of Cbl in cytoplasm occurs in a multiprotein complex composed of at least MS, MSR, MMACHC and MMADHC, which could contribute to shuttle safely and efficiently Cbl towards MS. Our data suggest that defective protein-protein interactions among key players of this pathway could contribute to the molecular mechanisms of the cblC, cblG and cblE genetic defects and provide novel insights into our understanding of the pathophysiology of inherited disorders of Cbl metabolism.