Objective: Rho-associated kinase (ROCK) is a serine/threonine kinase and a major downstream effector of the small GTP-binding protein, Rho. Rho-ROCK triggers an intracellular signaling cascade that controls actin cytoskeleton and is essential for cell motility and adhesion, neurite outgrowth and retraction. In chronic disabling disease, multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), demyelination and axonal damage are the major pathological changes contributing to neurological disability. We investigated the protective effect of a specific ROCK inhibitor, Y-39983, on demyelination and axonal damage in chronic EAE. Methods: Western blotting for myelin proteins, electron microscopy and solochrome cyanine staining was performed to evaluate demyelination while neurofilament proteins and cytoskeletal proteins including β-actin and β-tubulin were used to determine axonal damage in a chronic mouse model of EAE treated with Y-39983. Results: Y-39983 significantly suppressed clinical symptoms of EAE and prevented its relapse while increasing the amount of myelin proteins. No significant changes in neurofilaments and cytoskeletal proteins were observed compared with control EAE mice. The inhibition of demyelination by Y-39983 was confirmed by solochrome cyanine staining and electron microscopy. To further study the effect of Y-39983 on demyelination in EAE, we tested three major ROCK substrates, including myosin light chain phosphorylation, LIMK2 and collapsin response mediator protein-2. The activity of these molecules was decreased in EAE animals treated with Y-39983. Conclusion: The inhibitory effect of Y-39983 on demyelination is probably due to the inactivation of ROCK substrates, which are important for neurite outgrowth, growth cone collapse and demyelination of oligodendrocytes.