The efficacy of current anti-cancer gene therapies is limited by the inability of gene vectors to penetrate the poorly vascularized, hypoxic regions of tumors, leaving these sites untreated. We describe a new approach for targeting gene therapy to these sites, which employs an attenuated strain of the non-pathogenic bacterium, Salmonella typhimurium, carrying an exogenous (that is, reporter or therapeutic) gene under the regulation of a new, highly hypoxia-inducible promoter (FF+20(*)). This bacterial vector was seen to rapidly migrate into, and thrive in, hypoxic areas of both mammary tumor spheroids grown in vitro and orthotopic mammary tumors after systemic injection. Using the reporter gene construct, FF+20(*)-lacZ, we show that bacterial expression of high levels of beta-galactosidase occurred only in hypoxic/necrotic sites of spheroids and tumors. We then replaced the reporter gene with one encoding a novel cytotoxic protein (HlyE) and showed that this was also expressed by bacteria only in hypoxic regions of murine mammary tumors. This resulted in a marked increase in tumor necrosis and reduced tumor growth. Our system represents a promising new strategy for delivering gene therapy to poorly vascularized regions of tumors and shows, for the first time, the efficacy of HlyE as an anti-tumor agent.