Rickettsia rickettsii, a Gram-negative and obligate intracellular bacterium, preferentially infects the vascular endothelium during human infections leading to inflammation and dysfunction. The aim of this study was to determine whether R. rickettsii infection of endothelial cells (EC) activates p38 and/or c-jun N-terminal kinases (JNK) mitogen-activated protein (MAP) kinase, key regulatory proteins that control the response to inflammatory stimuli. We show that infection of cultured human EC results in the dose-dependent activation of p38, as assessed by increased phosphorylation and activity, without affecting the status of JNK. Rickettsia inactivation by heat or formaldehyde abolished the activation of p38 kinase and inhibition of cellular invasion by infection at low temperature, pre-treatment of host EC with cytochalasin D, or pre-incubation of rickettsiae with an irreversible phospholipase inhibitor led to a diminished p38 phosphorylation, suggesting requirement of invasion by viable rickettsiae for this host cell response. SB 203580, a p38-specific inhibitor, had no effect on infection-induced activation of the ubiquitous transcriptional regulator nuclear factor-kappa B, but effectively reduced the expression and secretion of important chemoattractant cytokines interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 by R. rickettsii-infected EC. Selective inhibition of p38 activity may be exploited as an anti-inflammatory target to prevent rickettsial vasculitis and to develop new and improved chemotherapeutic agents.