Scrub typhus is an acute febrile, mite-borne disease endemic to the Asia-Pacific region. In South Korea, it is a seasonal disease that occurs frequently in the autumn, and its incidence has increased steadily. In this study, we used a liquid chromatography and flow injection analysis-tandem mass spectrometry-based targeted urine metabolomics approach to evaluate the host response to Orientia tsutsugamushi infection. Balb/c mice were infected with O. tsutsugamushi Boryong, and their urine metabolite profile was examined. Metabolites that differed significantly between the experimental groups were identified using the Kruskal-Wallis test. Sixty-five differential metabolites were identified. The principal metabolite classes were acylcarnitines, glycerophospholipids, biogenic amines, and amino acids. An ingenuity pathway analysis revealed that several toxic (cardiotoxic, hepatotoxic, and nephrotoxic) metabolites are induced by scrub typhus infection. This is the first report of urinary metabolite biomarkers of scrub typhus infection and it enhances our understanding of the metabolic pathways involved.