Purpose: Proper aqueous humor (AH) dynamics is crucial for maintaining the intraocular pressure (IOP) in the eye. This study aims to investigate the function of Piezo2, a newly discovered mechanosensitive ion channel, in regulating AH dynamics. Methods: Immunohistochemistry (IHC) analysis and western blotting were performed to detect Piezo2 expression. The Cre-lox system was applied to create a conditional knockout model of Piezo2. IOP and aqueous humor outflow facility in live animals were recorded with a Tonometer and a syringe-pump system for up to 2 weeks. Results: We first detected Piezo2 with robust expression in the human trabecular meshwork (TM), Schlemm's canal (SC), the ciliary body's epithelium, and ciliary muscle. In addition, we found Piezo2 in human retinal ganglion cells (RGCs) and astrocytes in the optic nerve head (ONH). Through the Cre-lox system, Piezo2 can be successfully downregulated in mouse iridocorneal angle tissues. However, Piezo2 downregulation cannot significantly influence the IOP and outflow facility through the conventional pathway. Instead, we observed an effect of downregulated Piezo2 on decreasing the intercept in the flow rate versus pressure plot. According to the Goldmann equation, Piezo2 may function in regulating unconventional outflow, AH production, and episcleral venous pressure. Conclusions: These findings, for the first time, demonstrate that Piezo2 acts as an essential mechanosensor in maintaining the proper aqueous humor dynamics in the eye.