Dengue virus (DV) is a member of the family Flaviviridae. These positive strand RNA viruses encode a polyprotein that is processed in case of DV into 10 proteins. Although for most of these proteins distinct functions have been defined, this is less clear for the highly hydrophobic non-structural protein (NS) 4B. Despite its possible role as an antagonist of the interferon-induced antiviral response, this protein may play an additional more direct role for viral replication. In this study we determined the subcellular localization, membrane association, and membrane topology of DV NS4B. We found that NS4B resides primarily in cytoplasmic foci originating from the endoplasmic reticulum. NS4B colocalizes with NS3 and double-stranded RNA, an intermediate of viral replication, arguing that NS4B is part of the membrane-bound viral replication complex. Biochemical analysis revealed that NS4B is an integral membrane protein, and that its preceding 2K signal sequence is not required for this integration. We identified three membrane-spanning segments in the COOH-terminal part of NS4B that are sufficient to target a cytosolic marker protein to intracellular membranes. Furthermore, we established a membrane topology model of NS4B in which the NH2-terminal part of the protein is localized in the endoplasmic reticulum lumen, whereas the COOH-terminal part is composed of three trans-membrane domains with the COOH-terminal tail localized in the cytoplasm. This topology model provides a good starting point for a detailed investigation of the function of NS4B in the DV life cycle.