Columns are the morphological and functional units containing multiple neurons in the brain. The molecular mechanisms of column formation are largely unknown. Ephrin/Eph signaling mediates a variety of developmental processes. Ephrin acts as a ligand for Eph to regulate forward signaling, while Eph acts as a ligand for Ephrin to regulate reverse signaling. However, whether and how the uni- or bi-directional Ephrin/Eph signaling is involved in column formation remains elusive. In this study, we show that Ephrin and Eph regulate the morphology and location of columnar neurons through bi-directional repulsive signaling. Furthermore, Eph ligand triggers cytoplasmic tyrosine phosphorylation of Ephrin under the control of Src kinases and Fasciclin II (Fas2), forming Ephrin/Src/Fas2 complex to promote reverse signaling through a downstream regulator, Rac1. This study presents for the first time a unified picture of the molecular interactions in the defined context of column formation using the fly brain as a model.