Glaucoma is a widespread ocular disease characterized by a progressive loss of retinal ganglion cells (RGCs). Previous studies suggest that the cytokine tumor necrosis factor-α (TNF-α) may contribute to the disease process, although its role in vivo and its mechanism of action are unclear. To investigate pathophysiological mechanisms in glaucoma, we induced ocular hypertension (OH) in mice by angle closure via laser irradiation. This treatment resulted in a rapid upregulation of TNF-α, followed sequentially by microglial activation, loss of optic nerve oligodendrocytes, and delayed loss of RGCs. Intravitreal TNF-α injections in normal mice mimicked these effects. Conversely, an anti-TNF-α-neutralizing antibody or deleting the genes encoding TNF-α or its receptor, TNFR2, blocked the deleterious effects of OH. Deleting the CD11b/CD18 gene prevented microglial activation and also blocked the pathophysiological effects of OH. Thus TNF-α provides an essential, although indirect, link between OH and RGC loss in vivo. Blocking TNF-α signaling or inflammation, therefore, may be helpful in treating glaucoma.