AbstractThe Rab GTPase effector, Rab-coupling protein (RCP) is known to promote invasive behaviour in vitro by controlling integrin and receptor tyrosine kinase (RTK) trafficking, but how RCP influences metastasis in vivo is unclear. Here we identify an RTK of the Eph family, EphA2, to be a cargo of an RCP-regulated endocytic pathway which controls cell:cell repulsion and metastasis in vivo. Phosphorylation of RCP at Ser⁴³⁵ by Lemur tyrosine kinase-3 (LMTK3) and of EphA2 at Ser⁸⁹⁷ by Akt are both necessary to promote Rab14-dependent (and Rab11-independent) trafficking of EphA2 which generates cell:cell repulsion events that drive tumour cells apart. Genetic disruption of RCP or EphA2 opposes cell:cell repulsion and metastasis in an autochthonous mouse model of pancreatic adenocarcinoma—whereas conditional knockout of another RCP cargo, α5 integrin, does not suppress pancreatic cancer metastasis—indicating a role for RCP-dependent trafficking of an Eph receptor to drive tumour dissemination in vivo.