Migration and invasion enhancer 1 (MIEN1) is a novel gene found to be abundantly expressed in breast tumor tissues and functions as a critical regulator of tumor cell migration and invasion to promote systemic metastases. Previous studies have identified post-translational modifications by isoprenylation at the C-terminal tail of MIEN1 to favor its translocation to the inner leaflet of plasma membrane and its function as a membrane-bound adapter molecule. However, the exact molecular events at the membrane interface activating the MIEN1-driven tumor cell motility are vaguely understood. MIEN1 was first studied using in-silico analysis on available RNA sequencing data of human breast tissues and its expression was ascertained in breast cells. We performed several assays including co-immunoprecipitation, wound healing, western blotting and immunofluorescence to decipher the molecular events involved in MIEN1-mediated tumor cell migration. Clinically, MIEN1 is predominantly overexpressed in Her-2 and luminal B subtypes of breast tumors, and its increased expression correlates with poor disease free survival. Molecular studies identified a phosphorylation-dependent activation signal in the immunoreceptor tyrosine based activation motif (ITAM) of MIEN1 and the phosphorylation-deficient MIEN1-mutants (Y39F/50 F) to regulate filopodia generation, migration and invasion. We found that ITAM-phosphorylation of MIEN1 is significantly impaired in isoprenylation-deficient MIEN1 mutants indicating that prenylation of MIEN1 and membrane association is required for cross-phosphorylation of tyrosine residues. Furthermore, we identified MIEN1 as a novel interactor of Annexin A2 (AnxA2), a Ca2+ -dependent phospholipid binding protein, which serves as an extracellular proteolytic center regulating plasmin generation. Fluorescence resonance energy transfer (FRET) confirmed that MIEN1 physically interacts with AnxA2 and functional studies revealed that they mutually cooperate to accentuate tumor cell motility. Interestingly, our study identified that ectopic overexpression of MIEN1 significantly enhances Tyr23-phosphorylation on AnxA2, thereby stimulating cell surface translocation of AnxA2 and catalyzing the activation of its proteolytic activity. Our data show that the presence and interaction of both MIEN1 and AnxA2 in breast tumors are crucial drivers of cell motility. Our study has now deciphered a novel regulatory network governing the vicious process of breast tumor cell invasion-metastasis, and findings suggest MIEN1-AnxA2 as prospective targets to counter the deadly disease.