Salmonella enterica servovar Typhimurium is capable of targeting, colonizing, and eliciting growth suppression of tumors in mice. We examined the effects of mutations on this anticancer phenotype in two Salmonella virulence gene clusters. Salmonella pathogenicity island (SPI)-1 genes promote systemic invasion from the intestine, whereas SPI-2 genes support systemic survival within macrophages and other cells. Disabling SPI-1 (prgH(-)) strongly reduced invasion in vitro, but had no effect on tumor growth suppression in vivo. However, disabling SPI-2 (ssaT(-)) ablated tumor growth suppression. In addition to ssaT(-), mutations in SPI-2 genes sseA, sseB, sseC, sscA, and ssrA also eliminated antitumor activity, whereas mutations in sseF or sseG yielded partial loss of function. Impaired tumor amplification was seen in three SPI-2 mutants tested after intravenous or intratumoral injection. A SPI-2(-) strain was unable to suppress tumor growth in CD18-deficient mice with defective macrophages and neutrophils, suggesting that loss of tumor growth suppression in wild-type mice by SPI-2 mutants was not solely a function of increased susceptibility to immune attack. Thus, SPI-2 is essential for the Salmonella antitumor effects, perhaps by aiding bacterial amplification within tumors, and is the first identified genetic system for this Salmonella phenotype.