We identified a bovine B12 trafficking chaperone bCblC in Bos taurus that showed 88% amino acid sequence identity with a human homologue. The protein bCblC was purified from E. coli by over-expression of the encoding gene. bCblC bound cyanocobalamin (CNCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) in the base-off states and eliminated the upper axial ligands forming aquo/hydroxocobalamin (OH2/OHCbl) under aerobic conditions. A transition of OH2/OHCbl was induced upon binding to bCblC. Interestingly, bCblC-bound OH2/OHCbl did not react with reduced glutathione (GSH), while the reaction of free OH2/OHCbl with GSH resulted in the formation of glutathionylcobalamin (GSCbl) and glutathione disulfide (GSSG). Furthermore we found that bCblC eliminates the GSH ligand of GSCbl forming OH2/OHCbl. The results demonstrated that bCblC is a B12 trafficking chaperone that binds cobalamins and protects OH2/OHCbl from GSH, which could be oxidized to GSSG by free OH2/OHCbl. [BMB reports 2011; 44(3): 170-175]