Dengue virus (DENV) causes dengue fever, which is prevalent in the tropical and subtropical regions, and in recent years, has resulted in several major epidemics. Vimentin, a cytoskeletal component involved in DENV infection, is significantly reorganized during infection. However, the mechanism underlying the association between DENV infection and vimentin is still poorly understood. We generated vimentin-knockout (Vim-KO) human brain microvascular endothelial cells (HBMECs) and a Vim-KO SV129 suckling mouse model, combining the dynamic vimentin changes observed in vitro and differences in disease course in vivo, to clarify the role of vimentin in DENV-2 infection. We found that the phosphorylation and solubility of vimentin changed dynamically during DENV-2 infection of HBMECs, suggesting the regulation of vimentin by DENV-2 infection. The similar trends observed in the phosphorylation and solubility of vimentin showed that these characteristics are related. Compared with that in control cells, the DENV-2 viral load was significantly increased in Vim-KO HBMECs, and after DENV-2 infection, Vim-KO SV129 mice displayed more severe disease signs than wild-type SV129 mice, as well as higher viral loads in their serum and brain tissue, demonstrating that vimentin can inhibit DENV-2 infection. Moreover, Vim-KO SV129 mice had more disordered cerebral cortical nerve cells, confirming that Vim-KO mice were more susceptible to DENV-2 infection, which causes severe brain damage. The findings of our study help clarify the mechanism by which vimentin inhibits DENV-2 infection and provides guidance for antiviral treatment strategies for DENV infections.Copyright © 2022 Yu, Li, Zhou, Liu, He, Huang, Wu, Zhu, Yu, Yao, Zhang and Zhao.