Permeability alteration of microvascular endothelia is a factor in the plasma leakage produced by dengue virus (DV) infection, and beta3 integrin plays central roles in maintaining capillary integrity and regulating vascular permeability. In this study, interaction between beta3 integrin and DV serotype 2 (DV2) was investigated using human dermal microvascular endothelial cell line-1 (HMEC-1). We reported that DV2 infection could induce high expression level of beta3 integrin, and the high fluorescence intensity of beta3 integrin antigen observed in HMEC-1 after infection showed high co-localization with DV antigen. Pre-incubation of the virus with soluble alphanubeta3 integrin could strongly inhibit DV2 entry. And about 90% of virus entry was inhibited when beta3 integrin expression level was down-regulated by RNA interference. Our data indicated that DV2 infection could induce up-regulating expression of beta3 integrin, and beta3 integrin was required for DV2 entry into HMEC-1.