Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes cytokine-mediated alterations in the barrier function of the microvascular endothelium, leading to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We observed that DENV (serotype 2) productively infects primary (HMVEC-d) and immortalized (HMEC-1) human dermal microvascular endothelial cells, despite the absence of well-described DENV receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the mannose receptor on the cell surface. However, heparan sulfate proteoglycans (HSPGs) were highly expressed on these cells and pre-treatment of HMEC-1 cells with heparinase II or with glycosaminoglycans reduced DENV infectivity up to 90%, suggesting that DENV uses HSPGs as attachment receptor on microvascular endothelial cells. Sulfated Escherichia coli K5 derivatives, which are structurally similar to heparin/heparan sulfate but lack anticoagulant activity, were able to block DENV infection of HMEC-1 and HMVEC-d cells in the nanomolar range. The highly sulfated K5-OS(H) and K5-N,OS(H) inhibited virus attachment and subsequent entry into microvascular endothelial cells by interacting with the viral envelope (E) protein, as shown by surface plasmon resonance (SPR) analysis using the receptor-binding domain III of the E protein.