It is now widely recognized that the cross-talk between cancer and stromal cells may play a crucial role in cancer progression. However, little is known about the complex underlying molecular mechanisms that occur within the tumor microenvironment. Fibroblasts are the major stromal cells with multiple roles, especially toward both the extracellular matrix and the neighboring cell population, including neoplastic cells. Consequently, proteomic analyses would provide a wider resource for a better understanding of the potential modulating effects exerted by fibroblasts on cancer cells. In this article we describe the effects of fibroblast stimulation on the breast cancer cell line (8701-BC) proteomics, using a trans-well coculture system. Our results clearly indicate that fibroblasts induce considerable proteomic modulations on 8701-BC, mainly in the cytoskeleton proteins and glycolytic enzymes. Additionally, fibroblast-conditioned medium increased neoplastic cell proliferation and invasion with a concurrent upregulation of the c-Myc oncogene. Collectively these results suggest that fibroblast stimulation may enhance the malignant potential of breast cancer cells in vitro.