Rickettsia monacensis, a spotted fever group rickettsia, was isolated from Ixodes nipponensis ticks collected from live-captured small mammals in South Jeolla province, Korea in 2006. Homogenates of tick tissues were inoculated into L929 and Vero cell monolayers using shell vial assays. After several passages, Giemsa staining revealed rickettsia-like organisms in the inoculated Vero cells, but not the L929 cells. Sequencing analysis revealed that the ompA-small part (25–614 bp region), ompA-large part (2849–4455 bp region), nearly full-length ompB (58–4889 bp region) and gltA (196–1236 bp region) of the isolates had similarities of 100%, 99.8%, 99.3% and 99.5%, respectively, to those of R. monacensis. Furthermore, phylogenetic analysis showed that the isolate was grouped into the cluster in the same way as R. monacensis in the trees of all genes examined. These results strongly suggest that the isolate is closely related to R. monacensis. As far as is known, this is the first report of isolation of R. monacensis from ticks in Korea.