Tembusu virus (TMUV) is a mosquito-borne avian flavivirus, which was first isolated from Culex mosquitoes in Malaysia in 1955. Since 2010, the outbreak of TMUV in duck farms along the southeast coastal areas of China has caused huge economic losses in duck industry. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3'UTR are largely unknown. Here, using Tembusu virus (TMUV),an avian MBFV as models, we observed that the duck-derived SLI of the 3'UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3'UTR in viral host switching and pathogenicity changes in mice. This study was published online on Microbiology Spectrum (IF = 9.043) owned by American Society for Microbiology (ASM), and titled as "Stem-Loop I of the Tembusu Virus 3'-Untranslated Region Is Responsible for Viral Host-Specific Adaptation and the Pathogenicity of the Virus in Mice". DOI:10.1128/spectrum.02449-22.