Dengue virus (DENV) causes dengue fever, a major health concern worldwide. We identified an amphipathic helix (AH) in the N-terminal region of the viral nonstructural protein 4A (NS4A). Disruption of its amphipathic nature using mutagenesis reduced homo-oligomerization and abolished viral replication. These data emphasize the significance of NS4A in the life cycle of the dengue virus and demarcate it as a target for the design of novel antiviral therapy.