Pyridoxine (vitamin B₆) protects neurons against neurotoxicity. An excessive release of glutamate is widely considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. We investigated whether pyridoxine affected glutamate release in rat cerebral cortex nerve terminals (synaptosomes). Pyridoxine inhibited the release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration-dependent. Inhibition of glutamate release by pyridoxine was prevented by the vesicular transporter inhibitor bafilomycin A1, or by chelating intraterminal Ca²⁺, but was insensitive to DL-threo-β-benzyl-oxyaspartate, a glutamate transporter inhibitor. Pyridoxine did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. Examination of the effect of pyridoxine on cytosolic [Ca²⁺] revealed that diminution of glutamate release could be attributed to a reduction in voltage-dependent Ca²⁺ influx. Consistent with this, the pyridoxine-mediated inhibition of glutamate release was completely prevented by blocking the N- and P/Q-type Ca²⁺ channels, but not by blocking intracellular Ca²⁺ release or Na⁺/Ca²⁺ exchange. Furthermore, the pyridoxine effect on 4-AP-evoked glutamate release was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I (GF109203X) or bisindolylmaleimide IX (Ro318220), and pyridoxine significantly decreased the 4-AP-induced phosphorylation of PKC, PKCα, and myristoylated alanine-rich C kinase substrate. Together, these results suggest that pyridoxine inhibits glutamate release from rat cortical synaptosomes, through the suppression of presynaptic voltage-dependent Ca²⁺ entry and PKC activity.