Glaucoma is a leading cause of irreversible blindness, which is characterized by characteristic optic atrophy and visual field defects. Elevated intraocular pressure(IOP)is a primary risk factor of glaucoma, while the main cause of elevated IOP lies in the increased aqueous outflow resistance in pathological trabecular meshwork(TM), which is the conventional outflow pathway of aqueous humor. Rho-associated protein kinase inhibitor(ROCKi)is the IOP-lowering drug that is directly acting on the TM. The TM cell morphology, cell movement, cytokinesis and cell contraction by alteration of cytoskeleton can be changed by ROCKi to increase aqueous humor outflow facility and decrease IOP. ROCKi is now approved for clinical use in the United States and Japan. Meanwhile, it might play a role in optic nerve protection through increasing retinal vascular perfusion and promoting optic nerve regeneration. In addition, it decreases the possibility of filtration bleb scarring. Therefore, ROCKi has become a new pharmacological option to treat glaucoma. This article reviews the Rho-Rho kinase signaling pathway, the mechanism of ROCKi and its clinical application.