Most cancer vaccines induce CTL responses to tumor-associated antigens (TAA). Killing of tumor cells occurs through TAA-specific CTL-mediated cytolysis. Here, we show that one preventive followed by two therapeutic immunizations with an attenuated Listeria monocytogenes (LM)-based vaccine eradicates all metastases and almost the entire primary tumor in the syngeneic, aggressive mouse breast tumor model 4T1. We provide strong evidence that this is due to the combined result of direct kill by Listeria infecting the tumor cells and by CTL responses against Listeria antigens. We showed by electron microscopy that LM expressing truncated listeriolysin O (LLO) and amino acid fragments 311 to 660 of TAA Mage-b (LM-LLO-Mage-b(311-660)) and the control strain LM-LLO infect tumor cells in vitro and in vivo. In vitro data indicate that tumor cell death occurs through activation of NADP(+) oxidase and increased intracellular Ca(2+) levels, both resulting in the production of high ROS levels. Because both LM-LLO and LM-LLO-Mage-b(311-660) showed equally strong efficacies in vivo, we concluded that LM-LLO was crucial and Mage-b was of less importance. We found strong CTL responses to LM-LLO in the spleen, and depletion of CD8 T cells in vivo resulted in significant tumor regrowth (52%) in LM-LLO-vaccinated mice, indicating that LM-LLO-specific CTL indeed partially contributed to tumor cell kill in vivo. This dual mode of action of a Listeria-based vaccine has not been described before and may provide new directions in the development of more effective vaccines against metastatic breast cancer.