Glaucoma, a major cause of blindness, is characterized by elevated intraocular pressure (IOP) due to improper drainage of aqueous humor via the trabecular meshwork (TM) outflow pathway. Our recent work identified that loss of clusterin resulted in elevated IOP. This study delves deeper to elucidate the role of clusterin in IOP regulation. Employing an ex vivo human anterior segment perfusion model, we established that constitutive expression and secretion as well as exogenous addition of clusterin can significantly lower IOP. Interestingly, clusterin significantly lowered transforming growth factor β2 (TGFβ2)-induced IOP elevation. This effect was linked to the suppression of extracellular matrix (ECM) deposition and, highlighting the crucial role of clusterin in maintaining ECM equilibrium. A comprehensive global proteomic approach revealed the broad impact of clusterin on TM cell structure and function by identifying alterations in protein expression related to cytoskeletal organization, protein processing, and cellular mechanics, following clusterin induction. These findings underscore the beneficial modulation of TM cell structure and functionality by clusterin. Specifically, clusterin influences the actin-cytoskeleton and focal adhesion dynamics, which are instrumental in cell contractility and adhesion processes. Additionally, it suppresses the activity of proteins critical in TGFβ2, G-protein, and JAK-STAT signaling pathways, which are vital for the regulation of ocular pressure. By delineating these targeted effects of clusterin within the TM outflow pathway, our findings pave the way for novel treatment strategies aimed at mitigating the progression of ocular hypertension and glaucoma through targeted molecular interventions.