Loss of E-cadherin elicits epithelial-mesenchymal transition (EMT). While both the Src family of membrane-associated non-receptor tyrosine kinases (SFKs) and Slit2 binding to Roundabout 1 (Robo1) have been shown to induce E-cadherin repression and EMT, whether these two signaling pathways are mechanistically coupled remains unknown in epithelial cells. Here we found that Slit2 and Robol overexpression activated Src kinases for tyrosine phosphorylation, degradation of E-cadherin and induction of EMT. Specific blockade of Slit2 binding to Robol inactivated Src, prevented E-cadherin phosphorylation and EMT induction. Biochemically, the cytoplasmic CC3 motif of Robol (CC3) bound directly to the SH2 and 3 domains of c-Src and the cytoplasmic domains of E-cadherin. Slit2 induced Robol association with endogenous c-Src and E-cadherin, whereas ectopic expression of CC3 dissociated this protein complex in colorectal epithelial cells. These results indicate that Slit2 not only induces Robol binding to Src, but also recruits Src to E-cadherin for tyrosine phosphorylation of E-cadherin, leading to E-cadherin degradation and EMT induction in colorectal epithelial cells. (C) 2019 Elsevier Inc. All rights reserved.