Purpose: The purpose of this study was to investigate correlations of partial pressure of oxygen (pO2)in the ocular anterior segment of human eyes and aqueous humor antioxidant levels of ascorbate (AsA)and total reactive antioxidant potential (TRAP)with glaucoma and vitreous status. Methods: This prospective, cross-sectional study stratified patients (n = 288 eyes)by lens and vitreous status and the presence of primary open-angle glaucoma for statistical analyses. Intraocular pO2 concentrations were measured using a fiberoptic probe in patients at the beginning of planned glaucoma and/or cataract surgery. Aqueous humor specimens were obtained for antioxidant analysis of AsA and TRAP. Results: Following prior pars plana vitrectomy, pO2 levels were significantly higher than in the reference group of cataract surgery in the anterior chamber angle (16.2 ± 5.0 vs. 13.0 ± 3.9 mm Hg; P =.0171)and in the posterior chamber (7.6 ± 3.1 vs. 3.9 ± 2.7 mm Hg; P <.0001). AsA and TRAP levels were significantly lower (1.1 ± 0.4 vs. 1.4 ± 0.5 mM, respectively; 403.3 ±116.5 vs. 479.0 ± 146.7 Trolox units, respectively; P =.004 and P =.024, respectively)in patients after vitrectomy. In patients with an intact vitreous, neither pO2 nor antioxidant status correlated with lens status or glaucoma. Conclusions: Increased pO2 and antioxidant depletion following vitrectomy suggests an alteration of the intraocular oxidant-antioxidant balance. Our study links physiologic factors such as increased pO2 in the anterior chamber angle and the posterior chamber to decreased antioxidant levels in aqueous humor following vitrectomy. Oxidative stress/damage to the trabecular meshwork in such post-vitrectomy cases may contribute to intraocular pressure elevation and increased risk of glaucoma.