Orientia tsutsugamushi,an obligatory intracellular bacterium, is the causative agent of scrub typhus. Here the role of MAPK in TNF-α production in macrophages after infection withO. tsutsugamushihas been investigated. ERK1/2, JNK, and p38 MAPK became phosphorylated inOrientia-stimulated macrophages. Selective inhibitors of MAPK cascades could all significantly reduceOrientia-stimulated TNF-α production.Orientia-stimulated TNF-α production via p38 and JNK pathways was regulated by a post-transcriptional mechanism, whereas the ERK pathway mainly controlled the transcriptional step of TNF-α gene expression during infection. In conclusion, our data indicate that MAPK signaling is required to induce maximal TNF-α production in macrophages duringOrientiainfection.