Flaviviruses are enveloped, +ssRNA virus from the Flaviviridae family causing infection in North America, Oceania, Africa, Europe, the Middle east and west and central Asia It is well established that these viruses manipulate ER homeostasis and distribution to facilitate efficient replication. Due to its’ small genome size, flaviviruses utilise host factors to facilitate almost every stage of viral replication. Previous studies have shown that a ER membrane-shaping protein; Reticulon 3A (RTN3A), plays a crucial role in +ssRNA virus replication by inducing positive-membrane curvature (Diaz et al., 2012). In this study, we have observed that RTN3A is redistributed and recruited to the replication complex (RC) of West Nile virus strain Kunjin virus (WNVKUN) and Dengue virus strain New Guinea C (DENVNGC) in a neuroblastoma cell line. Subsequent analysis, by immunofluorescence (IF) and Fluorescence Resonance Energy Transfer (FRET), revealed that RTN3A interacts with the viral protein NS4A, known to be responsible for host membrane rearrangements in WNVKUN and DENVNGC infected cells. Via deletion mutagenesis we mapped the interaction between NS4A and RTN3A to the third transmembrane region as removal of the third transmembrane region reduced NS4A – RTN3A interaction by both IF analysis and pull-down experiments. Furthermore, siRNA-mediated knockdown of RTN3A attenuated WNVKUN viral replication, and severely affected the stability and expression of NS4A. In contrast, overexpression of RTN3A promoted oligomerisation of NS4A potentially a more stable form of this protein. Thus we have demonstrated the importance of RTN3A during flavivirus replication by interacting with, and stabilizing the NS4A protein.