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