It has been recently reported that vitamin K₂ (menaquinone-4: menatetrenone, VK₂) has an anti-atherogenic effect as well as the ability to produce clotting factors and improve osteoporosis. However, the mechanism by which VK₂ acts on atherosclerosis is still unclear. In this paper, we investigated the effects of vitamin K and its side chain on NO production as an anti-atherogenic substance in a cultured vascular system. Treatment of bovine vascular smooth muscle cells (SMC) with VK₂ (30μM) caused a time-dependent (24-72h) increase in the nitrite (NO^-₂) level in the conditioned medium, but not in bovine vascular endothelial cells. Classical NOS inhibitor (L-nitro arginine) and iNOS-specific inhibitors completely blocked the increased nitrite level induced by VK₂ treatment, but D-nitro arginine could not it. Immunostaining and western blotting analysis showed that VK₂ induced iNOS protein in the SMC. VK₂ has a naphtoquinone nucleus, which is identical in menadione (VK₃), and an unsaturated side chain, which is called geranylgeraniol (GGO). To determine whether the structure of VK₂ was related to an increasing nitrite level, we investigated the nitrite level in conditioned medium treated with VK₃ or GGO. Neither VK₃ nor GGO treatment of SMC increased the nitrite level. In addition, warfarin, an inhibitor of VK₂-dependent y-carboxylation, did not affect the increased nitrite level induced by VK₂ in SMC. In conclusion, VK₂ caused NO production through iNOS induction in bovine SMC, that was not related to the structure of VK₂, naphtoquinone nucleus or its side chain, independently of γ-carboxylation.