In addition to the functions of transporting melanosome in melanocytes and releasing contents of lytic granules in CTLs, Rab27A was recently shown to be involved in exocytosis of insulin and chromaffin granules in endocrine cells; it was also reported to be expressed in an exceptionally broad range of specialized secretory cells. As autocrine and paracrine cytokines are essential for invasion and metastasis in some solid tumors, blocking them may be an effective strategy to prevent tumor dissemination. In the present study, we show that Rab27A is associated with invasive and metastatic potentials of human breast cancer cells. The overexpression of Rab27A protein redistributed the cell cycle and increased the invasive and metastatic abilities in breast cancer cells both in vitro and in vivo. We also certified that Rab27A conferred the invasive and metastatic phenotypes on breast cancer cells by promoting the secretion of insulin-like growth factor-II (IGF-II), which regulates the expression of p16, vascular endothelial growth factor, matrix metalloproteinase-9, cathepsin D, cyclin D1, and urokinase-type plasminogen activator. These data provide functional evidence that Rab27A acts as a novel mediator of invasion and metastasis promotion in human breast cancer cells, at least in part, through regulating the secretion of IGF-II, suggesting that synergistic suppression of Rab27A and IGF-II activities holds a promise for preventing breast cancer invasion and metastasis.