Increased intraocular pressure (IOP) is the leading risk factor for glaucoma, but the mechanisms of IOP regulation during normalcy and disease are poorly understood. Considerable evidence suggests that Schlemm's canal (SC) endothelial cells may influence IOP by regulating aqueous humor outflow via the formation of trans-endothelial pores. This study employs a biomimetic perfusion system to explore pore formation in SC cells in vitro. Our results show that pore formation increases with increasing pressure drop, occurs only when flow is directed basal-to-apical across the cell layer, and it is reduced in glaucomatous versus normal SC cell lines. These results suggest that pore formation is a biomechanically regulated process and they establish our system as the first in vitro model that captures a specific pathology associated with glaucoma.