Dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV) are several important flaviviruses, and infections caused by these flaviviruses remain worldwide health problems. Different flaviviruses exhibit different biological characteristics and pathogenicity. Metabolomics is an emerging research perspective to uncover and observe the pathogenesis of certain infections.To improve the understanding of the specific metabolic changes that occur during infection with different flaviviruses, considering the principle of noninvasive sampling, this article describes our comprehensive analysis of metabolites in urine samples from the three kinds of flavivirus-infected mice using a liquid chromatography tandem mass spectrometry method to better understand their infection mechanisms.The urine of DENV-, JEV-, and ZIKV-infected mice had 68, 64, and 47 different differential metabolites, respectively, compared with the urine of control mice. Among the metabolic pathways designed by these metabolites, ABC transporters, arginine and proline metabolism, and regulation of lipolysis play an important role. Furthermore, we predicted and fitted potential relationships between metabolites and pathways.These virus-specific altered metabolites may be associated with their unique biological properties and pathogenicity. The metabolomic analysis of urine is very important for the analysis of flavivirus infection.Copyright © 2022 Xiaoyan Zheng and Ran Wang.