The Eph receptors are transmembrane proteins that belong to the receptor tyrosine kinases superfamily. Elevated Eph/ephrin expression levels have been associated with angiogenesis and tumor vasculature in many types of human cancers, including breast, lung, and prostate cancers, melanoma, and leukemia. In glioblastoma (GBM), the dysregulated expression of Eph receptors and of corresponding ephrin ligands has been associated with higher tumor grade and poor prognosis making them effective targets for therapeutic drugs. In this study, we describe the GL43.T, an anti-Eph aptamer, able to bind at high-affinity EphB3 and EphB2. Moreover, the GL43.T aptamer inhibits the glioma cell vitality and interferes with ephrine-B1 inhibition of chemotactic serum-stimulated cell migration. GL43.T aptamer represents a promising therapeutic molecule for EphB3-dependent cancers.