We have investigated motility in breast cancer cell lines in association with the expression of Transglutaminase type 2 (TG2) as well as upon the administration of Doxorubicin (Dox), an active cytotoxic agent that is employed in chemotherapy. The exposure of MCF-7 cells to the drug increased TG2 levels, triggering epithelial-mesenchymal transition (EMT), thereby supporting cell motility. The effects of Dox on the movement of MCF-7 cells were counteracted by treatment with NC9, a TG2 inhibitor, which induced morphological changes and also reduced the migration of MDA-MB-231 cells exhibiting high levels of TG2. The physical association of TG2 with the cytoskeletal component vimentin appeared pivotal both in drug-treated MCF-7 and in MDA-MB-231 cells and seemed to be independent of the catalytic activity of TG2. NC9 altered the subcellular distribution of TG2 and, consequently, the co-localization of TG2 with vimentin. Furthermore, NC9 induced a nuclear accumulation of TG2 as a prelude to TG2-dependent gene expression modifications. Since enzyme activity can affect both motility and nuclear functions, targeting of this protein could represent a method to improve therapeutic interventions in breast tumors, particularly those to control progression and to limit drug resistance.