Many essential proteins require pyridoxal 5’-phosphate (PLP), the active form of vitamin B6, as a cofactor for activity including enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian PLP-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial PLP levels in mammals remains unknown. Here, we used a genome-wide CRISPRi screen in erythroleukemia cells and organellar metabolomics to identify SLC25A38, a mitochondrial inner membrane protein, as a novel regulator of mitochondrial PLP. SLC25A38 loss causes depletion of mitochondrial, but not cellular, PLP, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial PLP-dependent enzymatic reactions, including serine to glycine conversion catalyzed by SHMT2 as well as ornithine aminotransferase (OAT). The proliferation defect of SLC25A38-null K562 cells in physiological and low B6 media can be explained by loss of SHMT2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial PLP maintenance and provides insights into the pathology of congenital sideroblastic anemia