An attenuated (ΔcyA, Δcrp) strain of Salmonella typhimurium (χ4550) containing a gene for human IL-2 (χ4550pIL2) reduces hepatic tumor burden when orally inoculated into mice with liver cancer; however, wild-type S. typhimurium is also associated with cancer regression. Therefore, experiments were designed to clarify the invasiveness and the anti-tumor properties of three strains of S. typhimurium. S. typhimurium χ4550pIL2, χ4550, or wild type (WT) was incubated with mature Caco-2 and HT-29 enterocytes, and S. typhimurium internalization was assessed. For infectivity experiments, mice were orally inoculated with saline or 109 S. typhimurium χ4550pIL2, χ4550, or WT; 48 h later mice were sacrificed for analysis of cecal bacteria and S. typhimurium translocation to mesenteric lymph nodes. For experiments involving tumor implantation, four groups were studied: saline control, tumor alone, χ4550pIL2+tumor, and χ4550+tumor. Mice were orally inoculated with saline or S. typhimurium and underwent laparotomy 24 h later with 5 × 104 MCA38 murine adenocarcinoma cells injected into the spleen. On day 14, liver tumors were counted and peripheral blood and hepatic lymphocyte populations were analyzed by FACScan. Attenuated S. typhimurium exhibited decreased internalization by cultured enterocytes and decreased infectivity after oral inoculation. Mice treated with χ4550pIL2 or χ4550 had fewer liver tumors and increased populations of hepatic and circulating NK1.1+CD3- lymphocytes compared to mice treated with saline (P < 0.01). These data suggest that attenuated S. typhimurium may have an application as an anti-tumor agent.