Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates in the cytosol of host cells. Although several protein antigens have been characterized and cloned, little information exists regarding the polysaccharide antigen of this bacterium. In this study, we characterized two monoclonal antibodies, NT19 and WT14, against the proteinase K-resistant antigen of O. tsutsugamushi. Western blot analysis showed that MAb NT19 and WT14 strongly recognized two antigenic bands with molecular masses of 20 kDa and 24 kDa, which were resistant to proteinase K digestion. We suggest that the proteinase-resistant antigen might be polysaccharide. One patient serum reacted with a 24 kDa band that was similar to a band observed by WT14, suggesting the possibility of the role of this proteinase-resistant antigen as an antigenic molecule in human infection.