Dengue virus (DENV) infection is one of the global arboviral epidemic; most common in the South East Asia. The severity of DENV infection ranges from dengue fever (DF), dengue hemorrhagic fever (DHF) to the most severe dengue shock syndrome (DSS). Plasma leakage and vascular permeability was evident in DHF/DSS, with marked symptoms of endothelial dysfunction. During DENV infection, cytokine and chemokine responses were reported to regulate the functional cytoskeleton protein networking, however, the exact role of individual cytoskeleton proteins is still unclear. The pro-inflammatory cytokine, Tumor Necrosis Factor-a (TNF-a) which was markedly elevated in patients with DHF/DSS, we recently identified the synergistic effect of DENV infection with TNF-a treatment causing the trans-endothelial permeability in human endothelial cells (EA.hy926). In the present study, our aim was to characterize the role of cytoskeleton proteins, in DENV-infected human endothelial cells with TNF-a activation. Mass spectrometry analysis (GeLC-MS/MS) was used to characterize the cytoskeleton proteomes in the DENV-mediated endothelial dysfunction. A total of 450 altered cytoskeleton proteins was found; where moesin was the most remarkably reduced cytoskeleton protein. Western blot analysis and immunofluorescence staining was used to further characterize the functional role of moesin in DENV-induced endothelial dysfunction. Further studies identified the structural reorganization of other two cytoskeleton proteins, including F-actin and vinculin in DENV-induced endothelial dysfunction. Trans-endothelial electrical resistant (TEER) assay was applied to measure the vascular permeability in the DENV-infected endothelial cells with TNF-a induction; and, the results were correlated with the expressions of moesin, F-actin and vinculin. Our results suggest the reduced expression of moesin regulate the endothelial permeability in severe forms of DENV infection. In conclusion, we identified the pivotal role of cytoskeleton protein in an in vitro model of DENV-induced endothelial dysfunction; this would paves in identifying therapeutic strategies towards the severe forms of DENV infection.