West Nile virus (WNV) is a neurotropic flavivirus that poses a continued global health risk since its emergence in North America in 1999, however, no vaccine is currently available for humans. Microvesicles (MV) are a 0.1-1µm extracellular vesicles derived from the plasma membrane by ectocytosis. Although implicated in inflammatory disease, they are little studied in viral infection. We show for the first time that WNV-infected endothelial cells produce large quantities of MV independently of WNV-induced cytopathy. UV-inactivated WNV did not induce MV production, indicating that viral replication is essential for MV induction. While conditioned medium from infected cultures did not itself induce MV, antibody neutralisation studies showed that TNF, but not IFN-b, was required for MV synthesis. Transmission electron microscopy revealed that MV comprised a homogeneous 100 nm and heterogeneous, 0.2 to 1 mm diameter population, but neither contained WNV virions. MV binding to recipient cells induced NF-kB activation and was antiviral. MV-expressed MHC-I, ICAM-I, PECAM-I and MAdCAM-1, as well as WNV proteins, PrM, E, NS1 and NS5, were detectable in recipient cells. Bone marrow-derived macrophages competently acquired WNV-induced MV, co-localising with lysosomes and induced WNV-specific memory T cell expansion. Finally, mice immunised i.p. with WNV-induced MV developed no disease and were protected from lethal WNV challenge, in contrast to UV-inactivated or live WNV immunisation alone and MV donor MHC did not need to be matched to recipient MHC. WNV-induced MV are thus evidently highly immunogenic and may represent an effective novel approach for vaccine development.