We investigated the role of MAPK in IFN-beta gene expression in macrophages after infection with Orientia tsutsugamushi. ERK1/2 became phosphorylated in Orientia-stimulated macrophages. Selective inhibition of ERK1/2 and p38 MAPK could all significantly reduce Orientia-stimulated IFN-beta mRNA expression. Orientia inactivation by heat abolished IFN-beta mRNA induction only, whereas cytochalasin D treatment completely blocked both IFN-beta and chemokine expression, suggesting requirement of cellular internalization by viable bacteria for IFN-beta gene induction. In conclusion, our data indicate that MAPK pathways are required to induce maximal IFN-beta gene expression in macrophages during Orientia infection.