Dengue virus (DENV) infection can cause mild dengue fever or potentially lethat dengue hemorrhage fever and shock syndrome (DHF/DSS). Vascular leakage is one of the main characteristics of DHF/DSS. Degradation of endothelial glycocalyx can cause the release of syndecan-1 (CD138), which is a marker of vascular leakage. It is known that DENV nonstructural protein 1 (NS1) can stimulate endothelial cells to secrete macrophage migration inhibitory factor (MIF) and heparanase 1 (HPA1), leading to glycocalyx degradation and hyperpermeability. However, it is unclear whether NS1 can induce metalloproteinase 9 (MMP9) secretion, which is increased in dengue patients’ sera and contribute to glycocalyx degradation as well. In this study, we first found that the sera levels of MIF, HPA1, MMP9 and CD138 were all increased in the dengue patients’ sera. However, only serum levels of MIF and CD138 showed a positive correlation in DHF patients. In addition, the secretions of MIF, HPA1, MMP9 and CD138 were increased in mice after recombinant NS1 (rNS1) challenge, and rNS1-induced glycocalyx degradation and vascular leakage were blocked in the presence of MIF inhibitors or its antibodies. In vitro studies demonstrated that rNS1 not only could stimulate endothelial cells to secrete HPA1. It could also stimulate human immune cells to secrete MMP9, leading to endothelial glycocalyx degradation and hyperpermeability. Furthermore, both of NS1-induced HPA-1 and MMP-9 secretion was inhibited by MIF inhibitors or its antibodies. Taken together, our results suggest that targeting MIF may represent a new therapeutic approach for the treatment of DENV-induced vascular leakage.