Salmonella enterica, a gram-negative pathogen, causes a spectrum of human infections including enterocolitis and typhoid fever. We previously showed that Salmonella flagellin played a role in suppressing intestinal mucosal inflammation in a murine model of acute enterocolitis. In this study, we examined the role of flagellin in the typhoid-like systemic murine Salmonella infection by measuring bacterial proliferation, inflammation, leukocyte recruitment, and cellular apoptosis in Peyer's patches (PPs), mesenteric lymph node (MLN), and spleen. We found that relative to an isogenic wild-type (WT) strain, aflagellate Salmonella exhibited increased proliferation at 4 days postinfection in PPs and MLN but not spleen. The aflagellate mutant also elicited increased local and systemic secretion of inflammatory cytokines such as interleukin-1beta, gamma interferon, and tumor necrosis factor alpha and enhanced surface expression of ICAM-1 on macrophages and dendritic cells (DCs). Furthermore, the recruitment of macrophages and DCs in PPs and MLN, but not spleen, was enhanced upon infection with aflagellate Salmonella. The relative differences between WT and aflagellate Salmonella were highly attenuated in Toll-like receptor 5-deficient (TLR5(-/-)) mice, indicating involvement of TLR5-dependent signaling. Interestingly, infection with the aflagellate mutant also resulted in decreased levels of T-cell apoptosis in PPs relative to infection with WT Salmonella. We postulate that the initial lack of detection of the aflagellate mutant in the mucosa permits increased proliferation within the host and enhances inflammatory signaling in nonepithelial cell types, which subsequently promotes leukocyte recruitment. In contrast, lack of difference in any disease parameter measured in the spleen likely reflects that Salmonella expression of flagellin is downregulated in this organ. Thus, the characteristic inflammatory pathology of Salmonella infection occurs only in PPs and to a lesser extent in MLN during the initial phases of infection and these early responses are dependent on TLR5.