Abstract We have previously developed a tumor-targeting Salmonella typhimurium double-amino acid auxotrophic strain termed A1-R (Cancer Research 66, 7647-7652, 2006; Proc. Natl. Acad. Sci. USA 104, 10170-10174, 2007). In the present study, we evaluated the efficacy of A1-R against a nude mouse model of human liver cancer. GFP-expressing A1-R bacteria targeted, colonized, and replicated in the human liver tumor growing s.c. in transgenic nude mice expressing RFP as observed by fluorescence imaging on day 3 after i.v. injection of A1-R. Subsequently, the bacteria grew throughout most of the tumor by day 10 after i.v. injection. Bacterial growth within the tumor was observed by fluorescence imaging of the infected tumor tissue in transverse sections. For efficacy experiments, bacteria (5 × 107 CFU/100 μl) were inoculated i.v. in HepG2-bearing nude mice. Tumor growth was monitored by caliper measurement in two dimensions. Treatment with A1-R resulted in tumor shrinkage. The difference in tumor volume between the treated and control group was statistically significant. There were no obvious toxic effects on the host even with 5 × 107 A1-R injected i.v. The mice tolerated A1-R treatment and could survive as long as untreated mice. A1-R only replicated and persisted within the microenvironment of the tumor and not outside the tumor site. The results suggest the potential of bacterial therapy of liver cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 583.