It is well established that +ssRNA viruses manipulate cellular protein and lipid homeostasis and distribution to facilitate efficient replication. Flaviviruses are enveloped, positive-sensed single-stranded RNA viruses from the Flaviviridae family causing infection in North America, Oceania, Africa, Europe, the Middle East and west and central Asia. One of the hallmarks of Flavivirus infection is the remodeling of host membranes that incorporates both viral and host factors to facilitate almost every stage of viral replication. In this study, we identified a key role for the membrane bending host protein Reticulon 3.1 (RTN3.1A) during the replication cycle of three Flaviviruses: West Nile virus (WNV), Dengue virus (DENV) and Zika virus (ZIKV). We observed that during Flavivirus infection, RTN3.1A is redistributed and recruited to the viral replication complex. Critically, siRNA-mediated knockdown of RTN3.1A expression attenuated WNV, DENV and ZIKV viral replication, severely affected the stability and abundance of the NS4A protein. Intriguingly, our observed recruitment of RTN3.1A was directly facilitated by the WNV NS4A protein, but this was not observed for DENV or ZIKV NS4A. EM analysis revealed a significant alternation and reduction of viral membrane structures in the endoplasmic reticulum when RTN3.1A was depleted. These observations have identified a crucial role of RTN3.1A for the viral remodeling of host membranes during efficient Flavivirus replication and the stabilization of viral proteins within the endoplasmic reticulum.