Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage, which leads to impaired aqueous humor outflow. Here, we explore a novel molecular mechanism involved in glaucomatous TM damage. We investigated the role of an endogenous Toll-like receptor 4 (TLR4) ligand, fibronectin-EDA (FN-EDA), in TGFβ2-induced ocular hypertension in mice. We utilized transgenic mouse strains that either constitutively express only FN containing the EDA isoform or contain an EDA-null allele and express only FN lacking EDA, with or without a mutation in Tlr4, in our inducible mouse model of ocular hypertension by injection of Ad5.TGFβ2. IOP was measured over time and eyes accessed by immunohistochemistry for total FN and FN-EDA expression. Constitutively active EDA caused elevated IOP starting at 14 weeks of age. Ad5.TGFβ2 induced ocular hypertension in wildtype C57BL/6J mice and further amplified the IOP in constitutively active EDA mice. TLR4 null and EDA null mice blocked Ad5.TGFβ-induced ocular hypertension. Total FN and FN-EDA isoform expression increased in response to Ad5.TGFβ2. These data suggest that both TLR4 and FN-EDA contribute to TGFβ2 induced ocular hypertension.