Genetic and epigenetic events often negate the cytostatic function of transforming growth factor-β (TGF-β) in mammary epithelial cells (MEC), which ultimately enables malignant MECs to proliferate, invade, and metastasize when stimulated by TGF-β. The molecular mechanisms underlying this phenotypic conversion of TGF-β function during mammary tumorigenesis remain poorly defined. We previously established α_vβ₃ integrin and Src as essential mediators of mitogen-activated protein kinase (MAPK) activation, invasion, and epithelial-to-mesenchymal transition stimulated by TGF-β in normal and malignant MECs. Mechanistically, β₃ integrin interacted physically with the TGF-β type II receptor (TβR-II), leading to its tyrosine phosphorylation by Src and the initiation of oncogenic signaling by TGF-β. We now show herein that Src phosphorylated TβR-II on Y284 both in vitro and in vivo. Interestingly, although the expression of Y284F-TβR-II mutants in breast cancer cells had no effect on TGF-β stimulation of Smad2/3, this TβR-II mutant completely abrogated p38 MAPK activation by TGF-β. Accordingly, Src-mediated phosphorylation of Y284 coordinated the docking of the SH2 domains of growth factor receptor binding protein 2 (Grb2) and Src homology domain 2 containing (Shc) TβR-II, thereby associating these adapter proteins to MAPK activation by TGF-β. Importantly, Y284F-TβR-II mutants also abrogated breast cancer cell invasion induced by α_vβ₃ integrin and TGF-β as well as partially restored their cytostatic response to TGF-β. Our findings have identified a novel α_vβ₃ integrin/Src/Y284/TβR-II signaling axis that promotes oncogenic signaling by TGF-β in malignant MECs and suggest that antagonizing this signaling axis may one day prove beneficial in treating patients with metastatic breast cancers. [Cancer Res 2007;67(8):3752–8]