The tyrosine kinase Src is over-expressed in numerous human cancers and is associated with poor prognosis. While Src has been extensively studied, its contributions to collective cell migration in vivo remain incompletely understood. Here we show that Src42A, but not Src64, is required for the specification and migration of the border cells in the Drosophila ovary, a well-developed and genetically tractable in vivo cell migration model. We found active Src42A enriched at border cell/nurse cell interfaces, where E-cadherin is less abundant, and depleted from border cell/border cell and border cell/polar cell junctions where E-cadherin is more stable, whereas total Src42A protein co-localizes with E-cadherin. Over-expression of wild type Src42A mislocalized Src activity and prevented border cell migration. Constitutively active or kinase dead forms of Src42A also impeded border cells. These findings establish border cells as a model for investigating the mechanisms of action of Src in cooperative, collective, cell-on-cell migration in vivo.