AIM: To investigate the effects of a selective inhibitor of Rho-associated kinase (ROCK), Y-27632, on inbred Wuzhishan porcine corneal endothelial cells (PCECs) in vitro and in vivo studies. METHODS: Primary PCECs were trypsinized from Wuzhishan miniature porcine corneal tissues. The optimal concentration of Y-27632 on PCECs was determined through MTT and 5-ethynyl-2’-deoxyuridine (EdU)-labeling assays. Seven New Zealand rabbits were used as a corneal endothelial dysfunction model, and a PCECs suspension supplemented with Y-27632 was injected into the anterior chamber of the rabbits. The progression of rabbit corneal opacity and edema were observed by slit lamp examination. The rabbits were sacrificed, and rabbit globes were enucleated for trypan blue-alizarin red staining, hematoxylin-eosin staining, and immunofluorescence analysis. RESULTS: Administration of 100 μmol/L Y-27632 facilitated PCECs’ proliferation obviously. The rabbit corneas injected with PCECs suspension and 100 μmol/L Y-27632 were restored to transparency significantly after 14d. CONCLUSION: The 100 μmol/L Y-27632 treatment improves PCECs’ proliferation significantly. And our results suggest that Y-27632 and PCECs can be used to treat corneal endothelial dysfunction.