The identity of microvascular endothelial (MVE) mechanosensors that sense blood flow in response to mechanical and chemical stimuli and regulate vascular permeability in the retina is unknown. Taking advantage of immunohistochemistry, calcium imaging, electrophysiology, impedance measurements and vascular permeability assays, we show that the transient receptor potential isoform 4 (TRPV4) plays a major role in Ca2+/cation signalling, cytoskeletal remodelling and barrier function in retinal microvasculature in vitro and in vivo. Human retinal MVECs (HrMVECs) predominantly expressed Trpv1 and Trpv4 transcripts, and TRPV4 was broadly localized to the plasma membrane of cultured cells and intact blood vessels in the inner retina. Treatment with the selective TRPV4 agonist GSK1016790A (GSK101) activated a nonselective cation current, robustly elevated [Ca2+]i and reversibly increased the permeability of MVEC monolayers. This was associated with disrupted organization of endothelial F‐actin, downregulated expression of occludin and remodelling of adherens contacts consisting of vascular endothelial cadherin (VE‐cadherin) and β−catenin. In vivo, GSK101 increased the permeability of retinal blood vessels in wild type, but not in TRPV4 knockout mice. Agonist‐evoked effects on barrier permeability and cytoskeletal reorganization were antagonized by the selective TRPV4 blocker HC 067047. Human choroidal endothelial cells (CECs) showed lower TRPV4 mRNA/protein levels and less pronounced agonist‐evoked calcium signals compared to MVECs. These findings demonstrate a major role for TRPV4 in Ca2+ homeostasis and barrier function in the human retinal microvascular endothelial barrier and suggest TRPV4 may differentially contribute to the inner vis à vis outer blood‐retinal barrier function. This article is protected by copyright. All rights reserved