Ripasudil hydrochloride hydrate (K-115), a specific Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, was the first ophthalmic solution developed for the treatment of glaucoma and ocular hypertension in Japan. Topical administration of K-115 decreased intraocular pressure (IOP) and increased outflow facility in rabbits. This study evaluated the effect of K-115 on monkey trabecular meshwork (TM) cells and Schlemm's canal endothelial (SCE) cells. K-115 induced retraction and rounding of cell bodies as well as disruption of actin bundles in TM cells. In SCE-cell monolayer permeability studies, K-115 significantly decreased transendothelial electrical resistance (TEER) and increased the transendothelial flux of FITC-dextran. Further, K-115 disrupted cellular localization of ZO-1 expression in SCE-cell monolayers. These results indicate that K-115 decreases IOP by increasing outflow facility in association with the modulation of TM cell behavior and SCE cell permeability in association with disruption of tight junction.