Prodigiosin (PG) is a red pigment produced by Serratia marcescens with pro-apoptotic activity in haematopoietic and gastrointestinal cancer cell lines, but no marked toxicity in non-malignant cells. Breast cancer is the most frequent malignancy among women in the European Union and better therapies are needed, especially for metastatic tumors. Moreover, multidrug resistance is a common phenomenon that appears during chemotherapy, necessitating more aggressive treatment as prognosis worsens. In this work, we extend our experiments on PG-induced apoptosis to breast cancer cells. PG was potently cytotoxic in both estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines. Cytochrome c release, activation of caspases-9, -8 and -7 and cleavage of poly (ADP-ribose) polymerase protein typified the apoptotic event and caspase inhibition revealed that PG acts via the mitochondrial pathway. In a multidrug-resistant subline of MCF-7 cells that over-expresses the breast cancer resistance protein, the cytotoxic activity of PG was slightly reduced. However, flow-cytometry analysis of PG accumulation and efflux in MCF-7 sublines showed that PG is not a substrate for this resistance protein. These results suggest that PG is an interesting and potent new pro-apoptotic agent for the treatment of breast cancer even when multidrug resistance transporter molecules are present.