Inborn errors of vitamin B₁₂ (cobalamin, Cbl) metabolism are rare Mendelian disorders that can present with elevations of methylmalonic acid and/or homocysteine in the blood and urine, and with hematological and neurological clinical findings. In mammalian cells, derivatives of Cbl are required as cofactors for two enzymes: adenosylcobalamin (AdoCbl) is required in the mitochondria as a cofactor to methylmalonyl CoA mutase (MCM); and methylcobalamin (MeCbl) is required in the cytoplasm as a cofactor to methionine synthase (MS). In the last two years, our laboratory has identified 13 patients with mutations in a transcriptional co-regulator HCFC1, and a single patient with mutations in its binding partner THAP11, in patients with a phenocopy of the cblC cobalamin inborn error. Recently a sixteen-month-old boy was referred to our clinical laboratory because of elevated serum methylmalonic acid and homocysteine levels. Additional clinical findings included cardiac defects (ventral septal defect, long QT syndrome), cleft palate, hypospadias, hyperbilirubinemia, intractable epilepsy, and global developmental delay. Cultured patient fibroblasts showed decreased function of both MCM and MS, and decreased synthesis of AdoCbl and MeCbl from exogenous [⁵⁷Co]cyanocobalamin. Crude subcellular fractionation demonstrated that most internalized radiolabelled Cbl was found in a fraction containing mitochondria, lysosomes and possibly other membrane-bound cellular compartments, unlike in control fibroblasts where it is found in the cytoplasmic fraction. Size exclusion chromatography of radiolabelled patient fibroblast extracts showed that the majority of labelled Cbl eluted bound to transcobalamin (TC), whereas the majority of label in control fibroblasts was present bound to MCM and MS. Whole exome sequencing did not identify causal mutations in any gene known to play a role in Cbl metabolism. Candidate causal variants were identified in the gene that codes for the transcription factor ZNF143, which has been shown to be part of a complex with HCFC1, and THAP11. Patient fibroblasts had decreased levels of ZNF143 protein. Immunofluorescence analysis showed an accumulation of lysosomes that co-localize with TC-Cbl in ZNF143 siRNA knockdown cells. There were also increased levels of lysosomal marker, Lamp1, in patient and ZNF143 knockdown cells. These data suggest that the cellular defect in that patient’s fibroblasts is affecting internalization of the TC-Cbl complex after binding to its cell surface receptor, but before degradation in the lysosome.