Previous work showed that ephrinA3/EphA4 forward signaling contributed to retinal ganglion cell (RGC) damage in experimental glaucoma. Since up-regulated patterns of ephrinA3 and EphA4 were observed in Müller cells and RGCs, an EphA4/ephrinA3 reverse signaling may exist in Müller cells of chronic ocular hypertension (COH) retina. We investigated effects of EphA4/ephrinA3 reverse signaling activation on Müller cells in COH retina. Intravitreal injection of the ephrinA3 agonist EphA4-Fc increased glial fibrillary acidic protein (GFAP) levels in normal retinas, suggestive of Müller cell gliosis, which was confirmed in purified cultured Müller cells treated with EphA4-Fc. These effects were mediated by intracellular STAT3 signaling pathway as phosphorylated STAT3 (p-STAT3) levels and ratios of p-STAT3/STAT3 were significantly increased in both COH retinas and EphA4-Fc intravitreally injected retinas, as well as in EphA4-Fc treated purified cultured Müller cells. The increase of GFAP protein levels in EphA4-Fc-injected retinas and EphA4-Fc treated purified cultured Müller cells could be partially eliminated by stattic, a selective STAT3 blocker. Co-immunoprecipitation results testified to the presence of interaction between ephrinA3 and STAT3/p-STAT3. In addition, intravitreal injection of EphA4-Fc or EphA4-Fc treatment of cultured Müller cells significantly up-regulated mRNA and protein contents of pro-inflammatory cytokines. Moreover, intravitreal injection of EphA4-Fc increased the number of apoptotic RGCs, which could be reversed by the tyrosine kinase blocker PP2. Overall, EphA4/ephrinA3 reverse signaling may induce Müller cell gliosis and increases release of pro-inflammatory factors, which could contribute to RGC death in glaucoma. Inhibition of EphA4/ephrinA3 signaling may provide an effective neuroprotection in glaucoma.