Inhalation of anthrax causes fatal bacteremia, indicating a meager host immune response. We previously showed that anthrax lethal toxin (LT) paralyzes neutrophils, a major component of innate immunity. Here, we have found that LT also inhibits actin-based motility of the intracellular pathogen Listeria monocytogenes. LT inhibition of actin assembly is mediated by blockade of Hsp27 phosphorylation, and can be reproduced by treating cells with the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580. Nonphosphorylated Hsp27 inhibits Listeria actin-based motility in cell extracts, and binds to and sequesters purified actin monomers. Phosphorylation of Hsp27 reverses these effects. RNA interference knockdown of Hsp27 blocks LT inhibition of Listeria actin-based motility. Rescue with wild-type Hsp27 accelerates Listeria speed in knockdown cells, whereas introduction of Hsp27 mutants incapable of phosphorylation or dephosphorylation causes slowing down. We propose that Hsp27 facilitates actin-based motility through a phosphorylation cycle that shuttles actin monomers to regions of new actin filament assembly. Our findings provide a previously unappreciated mechanism for LT virulence, and emphasize a central role for p38 MAP kinase-mediated phosphorylation of Hsp27 in actin-based motility and innate immunity.