To increase the potential of attenuated Salmonella as gene delivery vectors for cancer treatment, we developed a hypoxia-inducible promoter system to limit gene expression specifically to the tumor. This approach is envisaged to not only increase tumor specificity, but also to target those cells that are most resistant to conventional therapies. We demonstrate that the exponential growth of the attenuated bacteria is identical under normoxia and hypoxia. A hypoxia-inducible promoter (HIP-1) was created from a portion of the endogenous Salmonella pepT promoter and was shown to drive reporter gene expression under both acute and chronic hypoxia, but not under normoxia. Genetic engineering of the TATA- and FNR-box within HIP-1 allowed fine-tuning of gene induction, resulting in hypoxic induction factors of up to 200-fold. Finally, we demonstrate that HIP-1 can drive hypoxia-mediated gene expression in bacteria which have colonized human tumor xenografts in mouse models. Expression of both GFP and RFP under control of HIP-1 demonstrated an approximately 15-fold increase relative to a constitutive promoter when tumors were made hypoxic. Moreover, the use of a constitutive promoter resulted in reporter gene expression in both tumors and normal tissues, whereas reporter gene expressing using HIP-1 was confined to the tumor.