Glioblastoma is the most common brain cancer. Ephrins and their Eph receptors play important roles in the development of central nervous system and the regulation of cancer cell migration and invasion. In a search for the Eph receptor complexes, we used tandem affinity purification based interaction screening with tagged ephrins A1, A3 and A4 combined with protein identification by mass-spectrometry in U251MG glioblastoma cells. Ephrins bound to Eph receptors, mainly to EphA2 in these cells. Integrin α3 was identified in protein complexes with ephrin-As. Soluble ephrin-A1 colocalised with integrin α3 at the cell surface, and was rapidly endocytosed by the cells. However, integrin α3 did not colocalise with internalised ephrin-A1, whereas EphA2 receptor did. In U251MG cells, integrin α3 colocalised with EphA2 receptor at the cell edges and protrusions. Sites of EphA2-integrin α3 colocalisation were positive for vinculin, focal adhesion kinase and phosphotyrosine, that is, markers for cell adhesion and active signalling. The interaction between ephrin-As, Eph receptors and integrin α3 is plausibly important for the crosstalk between Eph and integrin signalling pathways at the membrane protrusions and in the migration of brain cancer cells.