A major risk for the development of primary open-angle glaucoma (POAG) is elevated intraocular pressure (IOP). Elevated IOP is caused by increased outflow resistance due in part to excessive extracellular matrix (ECM) deposition in the trabecular meshwork (TM). The role of transforming growth factor beta 2 (TGFβ2) in inducing ECM production is well understood. Recent studies suggest that toll-like receptor 4 (TLR4) plays an important role in fibrogenesis. We have previously described a crosstalk between TGFβ2 and TLR4 in the development of ocular hypertension and glaucomatous TM damage. Nuclear factor-kappa beta (NF-κB) is critical for TLR4 signaling. To determine the transactivation of NF-κB, TM cells were stimulated with cellular fibronectin containing the EDA isoform (cFN-EDA), TGFβ2, or lipopolysaccharide (LPS) in combination with a selective TLR4 inhibitor. cFN-EDA, TGFβ2, and LPS all induced transactivation of NF-κB and inhibition of TLR4 blocked the effect of each treatment paradigm. To evaluate the role of NF-κB in IOP regulation, we utilized our inducible mouse model of ocular hypertension by injection of Ad5.TGFβ2 in mice harboring a mutation in NF-κB and wild-type controls. IOP was measured over time and eyes accessed by immunohistochemistry for the ECM protein FN and the specific FN-EDA isoform. Ad5.TGFβ2 induced ocular hypertension and expression of FN and FN-EDA in wild-type mice, but mutation in NF-κB blocked the effect. These data suggest that NF-κB is necessary for TGFβ2-induced ECM production and ocular hypertension and the transactivation of NF-κB is dependent on both TGFβ2 and TLR4.