Flavivirus are enveloped viruses that enter into animal cells by fusion of viral and target membranes. The membrane fusion is triggered by the acidic pH inside the endosome and is mediated by glycoprotein E, a class II fusion protein. Both lipids and proteins play crucial and co-operative roles in the fusion process. Lipids contribute to the viral fusion process by their distinct chemical structure, composition and partitioning into specific microdomains in the plasma membrane. Here, we conducted a study on the requirement for specific lipids to viral fusion utilizing a liposomal model system (ANTS/DPX). Virus fusion events were measured fluorometrically using a content mixing fusion assay. Dengue virus fused efficiently with receptor-free liposomes consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion. Under optimal conditions, 30 to 40% of virus mediated fusion was obtained indicating a high mixing level, considering the viral particles (60 nm) and the liposomes size (100 nm). Our results show that Dengue virus fusion is absolutely dependent on the presence of cholesterol in the target membrane. However, the sphingomyelin does not seem to play an essencial role in the fusion process.