Transgelin is a protein which has been described as a marker of several cancers. Interestingly, the literature describes both up- and down-regulation of transgelin in the tumor in comparison with non-tumor tissue. The mechanisms of transgelin function in cancer are considerably unknown. Transgelin is abundant especially in smooth muscle cells. It is associated with actin stress fibers. These contractile structures participate among others in cell motility, adhesion or morphology. We focused on transgelin function in breast cancer. Initially, we studied transgelin role in cell migration in the breast cancer cell lines BT 549 and PMC 42. Using xCELLigence system we reached opposite results in two cell lines. Transgelin silencing increased and decreased migration of PMC 42 and BT 549 cells, respectively. To further clarify these contradictory results, we performed an experiment to quantify proteomic changes after transgelin silencing in these two cell lines using quantitative proteomics (iTRAQ-2DLC-MS/MS). Our results confirmed transgelin function in the migration of BT 549 cells and suggested transgelin role in apoptosis and small molecule biochemistry in PMC 42 cells.