The causes of cobalamin (B12, Cbl) deficiency are multifactorial. Whether nutritional due to poor dietary intake, or functional due to impairments in absorption or intracellular processing and trafficking events, the major symptoms of Cbl deficiency include megaloblastic anemia, neurological deterioration and in extreme cases, failure to thrive and death. The common biomarkers of Cbl deficiency (hyperhomocysteinemia and methylmalonic acidemia) are extremely valuable diagnostic indicators of the condition, but little is known about the changes that occur at the protein level. A mechanistic explanation bridging the physiological changes associated with functional B12 deficiency with its intracellular processers and carriers is lacking. In this article, we will cover the effects of B12 deficiency in a cblC-disrupted background (also referred to as MMACHC) as a model of functional Cbl deficiency. As will be shown, major protein changes involve the cytoskeleton, the neurological system as well as signaling and detoxification pathways. Supplementation of cultured MMACHC-mutant cells with hydroxocobalamin (HOCbl) failed to restore these variants to the normal phenotype, suggesting that a defective Cbl processing pathway produces irreversible changes at the protein level.