Vitamin K2 (VK2), which has been in wide use for the management of hypoprothrombinemia and osteoporosis in Japan, was tested for its efficacy on experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The severity of EAE was significantly ameliorated by the prophylactic administration of VK2, though it was not effective when given after the onset. Inflammatory cellular infiltration and the expression of both MHC class II and inducible nitric oxide synthase (iNOS) were reduced in the spinal cords of VK2-treated rats with EAE. The inhibitory effect of VK2 on the iNOS expression in glial cells was also observed in vitro. Considering the long use of VK2 without noticeable untoward effects, it may be applicable to the patients with MS.