We have developed a genetically-modified bacterial strain of Salmonella typhimurium, auxotrophic for leucine and arginine, which also expresses green fluorescent protein (GFP), termed S. typhimurium A1-R. S. typhimurium A1-R was found to be effective against metastatic human prostate, breast, and pancreatic cancer cell lines as well as osteosarcoma, fibrosarcoma and glioma cell lines in clinically-relevant nude mouse models. To understand the tumor-killing mechanism of S. typhimurium A1-R-GFP, we have studied their interaction with fluorescence-labeled cancer cells in vitro and in vivo. S. typhimurium-GFP invasion, proliferation, and virulence in the cancer cells were visualized by confocal fluorescence microscopy with the Olympus FV1000. We found that A1-R-induced cancer cell death had different mechanisms in different cancer cell lines. Some S. typhimurium A1-R-treated cancer cell lines became apoptotic and necrotic, and other cell lines burst after extensive intracellular bacterial growth. The time for S. typhimurium A1-R-GFP to kill the majority of the cancer cells varied from line to line, ranging from 2 hours to 72 hours. The understanding of the various mechanisms of cell killing by S. typhimurium A1-R will be important for its use as a general therapeutic for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2685. doi:1538-7445.AM2012-2685