Cancer cell migration and invasion play essential roles in the metastatic cascade that transforms the local, noninvasive confined tumor cells to the motile, metastatic cancer cells moving through the extracellular matrix and basement into the circulation. Accumulated evidences suggest that intratumoral hypoxia, a characteristic of fast-growing solid tumors, promotes cancer cell motility and invasive abilities. In this study, we investigated the effects of hypoxia and HIF-1α, surrounding fibroblasts, and p16 expression on the migration and invasion of breast cancer cells. We found that in general hypoxia promoted breast cancer cell migration and invasion, and cocultured fibroblasts stimulated invasiveness of breast cancer cells. Interestingly, HIF-1α from cancer cells and surrounding fibroblasts has respectively profound effects on the invasive ability of breast cancer cells. By applying both HIF-1α wild-type (WT) and knockout (KO) fibroblasts and breast cancer cells and using an in vitro invasion assay through commercially precoated invasion inserts, we found that fibroblasts, regardless their HIF-1α status, stimulated cell invasion of breast cancer cells that have intact HIF-1α. In contrast, only HIF-1α-expressing fibroblasts, not HIF-1α-nonexpressing ones, stimulated HIF-1α KO breast cancer cell invasion. These results suggest the complicated and respective HIF-1α contribution from breast cancer per se and surrounding fibroblasts to the tumor invasion and malignancy. Moreover, by using a Tet-on inducible system, we found that ectopic p16 gene transfer is capable of inhibiting hypoxia-induced cell migration and invasion of breast cancer cells, and suppressing cocultured fibroblast-stimulated invasiveness of breast cancer cells. These results suggest that p16, in addition to its well-known anti-tumor proliferation function, has novel anti-cancer properties capable of suppressing hypoxia/HIF-1α-mediated cancer cell migration and invasion. Furthermore, our current ongoing study implies an interaction between p16 and HIF-1α that may elucidate a novel p16-mediated tumor suppression pathway via HIF-1α-regulated signaling. These studies together may provide new information of the convergence of several important cell-growth regulation pathways on tumor invasion that would yield potential novel targets for effective cell- or gene-therapy strategy for treatment of breast cancer.