POAG is a progressive optic neuropathy that is currently the leading cause of irreversible blindness worldwide. While the underlying cause of POAG remains unclear, TGF-β2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Early studies have also demonstrated markedly elevated levels of oxidative stress markers in AH from POAG patients along with altered expression of antioxidant defenses. Here, using cultured primary or transformed human TM cells, we investigated the role oxidative stress plays at regulating TGF-β2-mediated remodeling of the ECM. Primary or transformed (GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-β2 and relative changes in intracellular reactive oxygen species (ROS) were measured using oxidation-sensitive fluorogenic dyes CellROX green or 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). TGF-β2-mediated changes in the content of connective tissue growth factor (CTGF) and collagen types 1α1 (COL1A1) and 4α1 (COL4A1) mRNA or collagens I and IV isoform proteins were determined in the absence or presence of mitochondrial-targeted antioxidants (XJB-5-131 or MitoQ) and quantified by quantitative PCR or by immunoblot and immunocytochemistry. Smad-dependent canonic signaling was determined by immunoblot, whereas Smad-dependent transcriptional activity was quantified using a Smad2/3-responsive SBE-luciferase reporter assay. Primary or transformed human TM cells cultured in the presence of TGF-β2 (5 ng/mL; 2 hours) exhibited marked increases in CellROX or fluorescein fluorescence. Consistent with previous reports, challenging cultured human TM cells with TGF-β2 elicited measurable increases in regulated Smad2/3 signaling as well as increases in CTGF, COL1A1, and COL4A1 mRNA and collagen protein content. Pretreating human TM cells with mitochondrial-targeted antioxidants XJB-5-131 (10 μM) or MitoQ (10 nM) attenuated TGF-β2-mediated changes in Smad-dependent transcriptional activity. The multifunctional profibrotic cytokine TGF-β2 elicits a marked increase in oxidative stress in human TM cells. Mitochondrial-targeted antioxidants attenuate TGF-β2-mediated changes in Smad-dependent transcriptional activity, including marked reductions in CTGF and collagen isoform gene and protein expression. These findings suggest that mitochondrial-targeted antioxidants, when delivered directly to the TM, exhibit potential as a novel strategy by which to slow the progression of TGF-β2-mediated remodeling of the ECM within the TM.