<p>Transient Receptor Potential Vanilloid 4 (TRPV4), a non-selective cation channel in trabecular meshwork (TM) tissue, is activated by forces like deformation and shear stress, playing a critical role in intraocular pressure (IOP) regulation. However, the mechanisms by which TRPV4 mediates TM cells responses to elevated intraocular pressure (IOP), along with the resulting regulatory outcomes, remain incompletely understood. In vitro, TM cells were mechanically stretched, and the effects on TRPV4 activation and calcium influx were evaluated via confocal microscopy, Western blotting, and immunofluorescence. Pharmacological agonists and inhibitors were used to investigate signaling mechanisms. In vivo, ocular hypertension (OHT) was induced in mice, and IOP was measured following drug treatments. Subsequently, IOP was measured after drug administration. Transcriptome sequencing was performed to detect TRPV4 activation-induced alterations in RNA expression and to explore associated regulatory pathways. Mechanical stretch significantly reduced <i>YAP1</i> mRNA expression in TM cells. TRPV4 activation induced YAP nuclear translocation, which was inhibited by HC067047, confirming TRPV4-YAP signaling. Transcriptome sequencing confirmed that the PI3K/AKT signaling pathway is crucial in mediating YAP activation. In vivo, HC067047 and VP treatment significantly lowered IOP and reduced the accumulation of ECM proteins in TM tissues. Our study demonstrates the critical role of TRPV4-YAP signaling in TM cell function and IOP regulation. These findings indicate that targeting the TRPV4-PI3K/AKT-YAP axis may offer novel therapeutic strategies for glaucoma.​</p>