PURPOSE. To investigate the roles of vasoactive intestinal peptides (VIPs) in regulating the morphology and F-actin distribution of Schlemm's canal (SC) of rat eyes. METHODS. Chronic intraocular pressure (10P) hypertension models with episcleral venous cauterization (EVC) were treated with topical VIP or PG99-465 (vasoactive intestinal peptide receptors 2 (VPAC21 antagonist). 10Ps were measured with Tono-Pen, and the SC parameters, including the cross-section area, circumference, and length, were statistically evaluated by hematoxylin-eosin and CD31 immunohistochemical staining. Immunofluorescence was performed to detect the distribution of F-actin in the SC. Moreover, the distribution of filamentous actin (F-actin) and globular actin (G-actin) in human umbilical vein endothelial cells (HUVEC5) was studied under a pressure system by immunofluorescence and Western blotting. RESULTS. Increased expressions of VIP and VPAC2 receptors, as well as a disordered distribution of F-actin were found in SC endothelial cells (SCEs) in the EVC model. Moreover, topical VIP maintained the normal distribution of F-actin in SCEs, expanded the collapsed SC, and induced a significant decrease in IOP in the EVC model. In in vitro HUVEC5, the F-actin/Gactin ratio increased significantly under stress stimulation for 30 minutes. A total of 50 mu M VIP helped maintain the normal F-actin/G-actin ratio of HUVEC5 against stress stimulation. CONCLUSIONS. VIP regulates the distribution of F-actin in SCEs via the VPAC2 receptor in order to induce a decrease in IOR VIP may represent a new target for antiglaucoma drugs.