To date we know of four inborn errors of autosomal recessive inheritance that lead to vitamin B6-dependent seizures. Among these, pyridoxine-dependent seizures due to antiquitin deficiency is by far the most common, although exact incidence data are lacking. In PNPO deficiency, samples have to be collected prior to treatment, while PDE, hyperprolinemia type II and congenital HPP can be diagnosed while on vitamin B6 supplementation. A vitamin B6 withdrawal for diagnostic purposes is nowadays only indicated in patients with a clear vitamin B6 response but normal biochemical work-up. In the presence of therapy-resistant neonatal seizures, early consideration of a vitamin B6 trial over 3 consecutive days is crucial in order to prevent irreversible brain damage. While PLP would be effective in all four disorders, pyridoxine fails to treat seizures in PNPO deficiency. As PLP is unlicensed within Europe and North America, pyridoxine is widely used as the first line drug, but if it is ineffective it should be followed by a trial with PLP, especially in neonates. As severe apnea has been described in responders, resuscitation equipment should be at hand during a first pyridoxine/PLP administration. Patients and parents have to be informed about the lifelong dependency and recurrence risks in forthcoming pregnancies.