PURPOSE. A previous study reported that vasoactive intestinal peptide (VIP) can regulate the cytoskeleton of Schlemm's canal (SC) endothelium and expand the SC lumen in a rat glaucoma model. In this study, we aimed to investigate the molecular mechanism of VIP on cytoskeleton regulation. METHODS. During in vivo experiments in rats, leucine-rich repeat kinase 2 (LRRK2) expression and the ratio of F-actin to G-actin (F-/G-actin) surrounding SC were examined by immunofluorescence after the application of VIP. For in vitro experiments in human umbilical vein endothelial cells, both quantitative PCR (qPCR) and western blotting were performed to evaluate Sp1 and LRRK2 expression after the application of VIP (and Sp1/LRRK2 inhibitor). In addition, the F-/G-actin ratio was examined by both immunofluorescence and western blotting after the application of VIP (and LRRK2 inhibitor). RESULTS. VIP induced increases in the expression of LRRK2 both in vivo and in vitro and the nuclear translocation of Sp1 in vitro. The application of Sp1 inhibitor abolished the increase in LRRK2 expression induced by VIP in vitro. In addition, VIP changed the F-/G-actin ratio, and this effect was abolished by the LRRK2 inhibitor both in vivo and in vitro. CONCLUSIONS. VIP increased the expression of LRRK2, and this regulation was due to the nuclear translocation of Sp1. VIP further changed the F-/G-actin ratio and regulated the balance between the stabilization and destabilization of the F-actin architecture. This study elucidates a novel mechanism by which VIP regulates the actin cytoskeleton of SC endothelium via the Sp1-LRRK2 pathway, suggesting a potential novel treatment strategy for glaucoma.