Eph receptors and their cognate ligands ephrins are important players in axon guidance and neural patterning during development of the nervous system. Much of our knowledge about the signal transduction pathways triggered by Eph receptors has been related to the modulation of actin cytoskeleton, which is fundamental in mediating the cellular responses in growth cone navigation, cell adhesion, and cell migration. In contrast, little was known about whether long term activation of Eph receptor would regulate gene expression. Here we report a novel signaling pathway of EphA4, which involves activation of the tyrosine kinase Jak2 and the transcriptional activator Stat3. Transfection of COS7 cells with EphA4, but not the kinase-dead mutant, induced tyrosine phosphorylation of Jak2, Stat1, and Stat3. Treatment of cultured C2C12 myotubes with ephrin-A1 also induced tyrosine phosphorylation of Stat3, which was abolished by the Jak2 inhibitor AG490. Moreover, Jak2 was co-immunoprecipitated with EphA4 in muscle, and both proteins were concentrated at the neuromuscular junction (NMJ) of adult muscle. By using microarray analysis, we have identified acetylcholinesterase, the critical enzyme that hydrolyzed the neurotransmitter acetylcholine at the NMJ, as a downstream target gene of the Jak/Stat pathway in muscle. More importantly, ephrin-A1 increased the expression of acetylcholinesterase protein in C2C12 myotubes, which was abolished by AG490. In contrast, ephrin-A1 reduced the expression of fibronectin mRNA in C2C12 myotubes independently of Jak2. Finally, the expression level of acetylcholinesterase in limb muscle of EphA4 null mice was significantly reduced compared with the wild-type control. Taken together, these results have identified Jak/Stat proteins as the novel downstream targets of EphA4 signaling. In addition, the present study provides the first demonstration of a potential function of Eph receptors and Jak/Stat proteins at the NMJ.