Flavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here we used dengue virus serotype-2 (DENV-2) as a model to characterize viral NS4A/NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimated Kd of 50 nM. Amino acids 40-76 (spanning the first transmembrane domain [amino acids 50-73]) of NS4A and amino acids 84-146 (also spanning the first transmembrane domain [amino acids 101-129]) of NS4B are the determinants for NS4A/NS4B interaction. NMR analysis suggests that NS4A residues17-80 form two amphipathic helices (helix α1 [residues 17-32] and helix α2 [residues 40-47]) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52-75) that transverses ER membrane. In addition, NMR analysis identified NS4A residues that may participate in NS4A/NS4B interaction. Amino acid-substitution of these NS4A residues exhibited distinct effect on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that abolished or severely reduced viral replication affected NS4A/NS4B interaction; in contrast, two NS4A mutations (F71A and G75A) that did not attenuate viral replication did not affect NS4A/NS4B interaction, demonstrating the biological relevance of NS4A/NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A/NS4B interaction could be a potential antiviral approach.