This work was funded by National Institute of Health Grants R01 EY016112, EY015266, and EY014801 and an unrestricted grant to the University of Miami's Bascom Palmer Eye Institute from Research to Prevent Blindness. Financial support from Fight for Sight is gratefully acknowledged. Funding to XG was provided by Instituto de Salud Carlos III, Spain (FIS PI14/00141 and RETIC RD12/0034/0003) and Generalitat de Catalunya (2014SGR1165). In the eye, intraocular pressure (IOP) is tightly regulated and its persistent increase leads to ocular hypertension and glaucoma. We have previously shown that trabecular meshwork (TM) cells might detect aqueous humor fluid shear stress via interaction of the extracellular matrix (ECM) protein cochlin with the cell surface bound and stretch-activated channel TREK-1. We provide evidence here that interaction between both proteins are involved in IOP regulation. Silencing of TREK-1 in mice prevents the previously demonstrated cochlin-overexpression mediated increase in IOP. Biochemical and electrophysiological experiments demonstrate that high shear stress-induced multimeric cochlin produces a qualitatively different interaction with TREK-1 compared to monomeric cochlin. Physiological concentrations of multimeric but not monomeric cochlin reduce TREK-1 current. Results presented here indicate that the interaction of TREK-1 and cochlin play an important role for maintaining IOP homeostasis.