Eph receptors, the largest subfamily of receptor tyrosine kinases, and their ephrin ligands play important roles in nervous system development. Recently, they have been implicated in tumorigenesis of different cancers. In this study, we showed that the expression of ephrinA5 was dramatically downregulated in primary gliomas compared with normal tissues. Forced expression of ephrinA5 reduced tumorigenicity of human glioma U373 cells. Epidermal growth factor receptor (EGFR), which frequently acts as an oncoprotein in glioma, was greatly decreased in ephrinA5-transfected glioma cells, and the two molecules exhibited a mutually exclusive expression pattern in primary glioma samples. We found that ephrinA5 enhanced c-Cbl binding to EGFR, thus promoted ubiquitylation and degradation of the receptor. Either ephrinA5-Fc or EphA2-Fc treatment simulating bidirectional signaling of Eph/ephrin system resulted in EGFR decrease. This study discovered that ephrinA5 acted as a tumor suppressor in glioma, and its negative regulation of EGFR contributed to the suppressive effects. In addition to identifying a novel mechanism underlying tumor suppressor activity of ephrinA5, we also showed cross-talk between different receptor tyrosine kinase families in glioma. These findings may improve therapeutic strategies for glioma.