Flagellin is the structural component of flagella produced by many pathogenic bacteria and is a potent proinflammatory molecule that mediates these effects through Toll-like receptor (TLR) 5. In Listeria monocytogenes (LM), flagellin expression is regulated by temperature and has been described as being shut off at 37°C. In this study, we demonstrate that TLR5-mediated cell activation and flagellin expression is maintained at 37°C in some laboratory-adapted strains and in ≈ 20% of LM clinical isolates. To determine the role of flagellin in LM infection, a targeted mutation in the structural gene for flagellin (flaA) was generated in a parental LM strain that expressed flagellin under all conditions examined. In vitro studies demonstrated that this ΔflaA mutant was (i) non-motile; (ii) not able to activate TLR5-transfected HeLa cells; and (iii) induced tumour necrosis factor (TNF)-α production in ≈ 50% fewer CD11b+ cells in splenocytes from normal mice compared with the parental strain. However, there was no significant alteration in virulence of the ΔflaA mutant after either intravenous or oral murine infection. Similarly, there was no difference in the generation of LM-specific CD8 or CD4 T cells after intravenous or oral infection. These data indicate that flagellin is not essential for LM pathogenesis or for the induction of LM-specific adaptive immune responses in normal mice.