The Ca2+-mediated signaling pathways are implicated either directly or indirectly in tumorigenesis and tumor progression. The store-operated Ca2+ entry (SOCE) is the major Ca2+ entry mechanism in non-excitable cells, which are most cancer cell origin. Ca2+ influx is required for serum- or growth factor-induced cell migrations. Moreover, growth factor signaling has been altered in most cancers. However, little is known about underlying mechanism of growth factor-mediated SOCE activation involved in cancer cell migration. Here, we examined the molecular mechanism of serum growth factor-mediated Orai1 activation in human breast cancer cell migration.Cell migration was specifically blocked by SOCE inhibitors such as SKF96365 and 2APB but not by voltage-gated calcium channel inhibitor, nimodipine. Orai1-induced cell migration was blunted by silencing of Orai1, whereas overexpression of Orai1 augmented SOCE. SOCE was increased by treatment of serum or diverse growth factors including EGF, IGF1 and PDGF. Serum-induced augmentation of SOCE and cell migration were blocked by treatment of PI3K inhibitors (Wortmannin and LY294002) or Erk1/2 inhibitor (U0126) indicating that both Akt and Erk1/2 signaling pathways are crucial for SOCE. Serum increased surface abundance of Orai1 channels, whereas activation kinetics of Orai1 channel was not altered. Brefeldin A and botulinium toxin interfering with vesicle associated trafficking reduced SOCE. Moreover, Orai1 physically interacts with vesicle-associated membrane protein-2 (VAMP2). SOCE was decreased by silencing of VAMP2 or by treatment with tetanus toxin supporting the notion that serum and growth factors increase cell surface abundance of Orai1 channels via activating VAMP2-dependent secretory pathway. These results provide a novel molecular basis for serum growth factor-mediated Ca2+ regulation in human breast cancer cell migration.