Permeability alteration of microvascular endothelia is a factor in the plasma leakage produced by dengue virus (DV) infection, and β3 integrin plays central roles in maintaining capillary integrity and regulating vascular permeability. In this study, interaction between β3 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 β3 integrin, and the high fluorescence intensity of β3 integrin antigen observed in HMEC-1 after infection showed high co-localization with DV antigen. Pre-incubation of the virus with soluble ανβ3 integrin could strongly inhibit DV2 entry. And about 90% of virus entry was inhibited when β3 integrin expression level was down-regulated by RNA interference. Our data indicated that DV2 infection could induce up-regulating expression of β3 integrin, and β3 integrin was required for DV2 entry into HMEC-1.