Oxidative stress and inflammation play a key role in pathophysiology of glaucoma. Baicalin is known as an anti-oxidative and anti-inflammatory substance, possessing the potential to treat glaucoma. This study was designed to assess the anti-oxidative and anti-inflammatory potentials in culture human trabecular meshwork (hTM) cells. Using hTM cells as the in vitro model, we investigated the effects of baicalin on oxidative stress-induced markers for hTM impairments. We pre-incubated hTM cells with baicalin before hydrogen peroxide stressing or pre- plus co-incubated with baicalin before and during stressing and monitored the cell death, production of intracellular reactive oxygen species (iROS) and inflammatory cytokines, accumulation of carbonylated proteins, and activity of senescence marker. Samples that received pre- plus co-treatment with 10 or 15 μM baicalin showed significantly increased cell survival and decreased iROS production. Further studies demonstrated that pre- plus co-treatment with 15 μM baicalin significantly inhibited proinflammatory factor IL-1α and ELAM-1 production, decreased activities of senescence marker SA-β-gal, and lowered carbonylated protein levels. In contrast, samples that received only pre-treatment did not show any of these protective effects. Baicalin can protect hTM cells against oxidative stress, shedding light on potential treatment for glaucoma.