Shiga toxin (Stx) causes hemolytic uremic syndrome. The cytotoxic effect of Stx is mediated by its receptor, globotriaosylceramide (Gb3), which has a limited expression profile in human cells but is overexpressed in many neoplastic cells, including breast cancer. Triple-negative breast cancer (TNBC) is the most aggressive and difficult to treat. On the other hand, TDP-43 is a key protein in the pathogenesis of several neurodegenerative diseases, and both TDP-43 aggregation and abnormal cytoplasmic mislocalization are critical features leading to neuronal degeneration. However, little is known about its role in other diseases, such as cancer. In this work we explore a) the potential of Stx as a novel cytotoxic agent in the human TNBC cell line MDA-MB-231, and b) the changes in TDP-43 elicited by these toxins. TNBC cells were treated with Stx1, Stx2, or anti-Gb3 antibody, while the non-tumorigenic mammary epithelial cell line NMuMG and VERO cells were used as negative and positive controls for Gb3 expression, respectively. Gb3 expression and Stx uptake were observed in MDA-MB-231 and VERO cells. MTT assay results showed that 10 ng/ml of Stx1, Stx2, and anti-Gb3 significantly reduced TNBC cell viability by 50%, 40%, and 10% respectively after 48 hours. Moreover, 10 ng/ml of these toxins significantly increased the number of cells with karyorrhexis and autophagy. In addition, these treatments reduced the percentage of mitotic cells, BrdU incorporation and cell migration rate. Pre-incubation with PPMP (a Gb3 synthesis inhibitor) significantly reversed all observed effects. Remarkably, Stx significantly increased both total TDP-43 expression and cytoplasmic mislocalization, while both parameters were significantly decreased when cells were also treated with PPMP. In summary, MDA-MB-231 cells are susceptible to Stx and anti-Gb3, suggesting that Stx could be used as an antineoplastic agent in TNBC. We also propose that TDP-43 may play a role in Stx2-mediated cytotoxicity.