Flavivirus genome encodes seven non-structural proteins (NSPs) and these NSPs are believed to be involved in their genomic RNA replication, of which the mechanism is unclear. We find that West Nile virus (WNV) NSPs were capable of self-assembling membranous vesicles in cells, which are composed of the host endoplasmic reticulum membrane integrated with viral NS1 and NS4A, and possibly NS2A. The vesicles can further organize into replication complex (RC)-associated vesicles which combine both the vesicle and predicted RC components. The authentic RC -associated vesicles were observed in cells transfected with infectious WNV cDNA as well as WNV replicon. Further mutational analysis showed that WNV/DENY heterologous NS polyproteins derived from lethal chimeric recombinants produced abnormal vesicles. Site-directed mutation of either NS2A or NS4A, which resulted in failure of viral RNA replication, caused immature vesicles too. These findings reveal molecular composition and assembly of the virus-specific nanomachine and confirm that these structures are used for the viral RNA replication.