Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4’-deoxypyridoxine (4dPN) is a long known B6antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organismEscherichia coliK12, we showed that 4dPN cannot be used as a source of vitamin B6as previously claimed and that it is toxic in several conditions where vitamin B6homeostasis is affected, such as in a B6auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene,yggS. In addition, we found that 4dPN sensitivity is likely the result of multiple modes of toxicity, including inhibition of PLP-dependent enzyme activity by 4’-deoxypyridoxine phosphate (4dPNP) and inhibition of cumulative pyridoxine (PN) uptake. These toxicities are largely dependent on the phosphorylation of 4dPN by pyridoxal kinase (PdxK).