Evidence indicates that 1 alpha, 25-dihydroxy vitamin D3 (1, 25-(OH)(2)D-3) markedly reduces intraocular pressure (IOP) in nonhuman primates, while the biochemical mechanisms are unclear. To investigate the influence of oxidative stress on human trabecular meshwork cells (HTMCs) and the effect and regulatory mechanism of 1, 25-(OH)(2)D-3 in HTMCs under oxidative stress, we established an oxidative stress model in HTMCs using hydrogen peroxide (H2O2) and showed that 1, 25-(OH)(2)D-3 could inhibit oxidative stress induced apoptosis and reduce extracellular matrix (ECM) composition of HTMCs. Moreover, 1, 25(OH)(2)D-3 could attenuate H2O2-induced inflammation in HTMCs. Mechanistically, our findings revealed that H2O2 -induced damage was mediated by the transforming growth factor -beta (TGF-beta)-SMAD3 pathway in HTMCs, and 1, 25-(OH)(2)D-3 could protect HTMCs against oxidative stress through vitamin D receptor (VDR), which antagonises the effects of SMAD3. Overall, these findings define a mechanism by which 1, 25-(OH)(2)D-3 reduces ECM accumulation and suppresses the TGE-beta-SMAD3-VDR pathway in HTMCs, thus protecting the cells from oxidative stress, suggesting 1, 25-(OH)(2)D-3 might be a potential therapeutic for glaucoma. (C) 2019 Elsevier Inc. All rights reserved.