Flaviviruses, such as the dengue virus and the West Nile virus (WNV), are arthropod-borne viruses that represent a global health problem. The flavivirus lifecycle is intimately connected to cellular lipids. Among the lipids co-opted by flaviviruses, we have focused on sphingomyelin, an important component of cellular membranes particularly enriched in the nervous system. After infection with the neurotropic WNV, mice deficient in acid sphingomyelinase (ASM), which accumulate high levels of sphingomyelin in their tissues, displayed exacerbated infection. In addition, WNV multiplication was enhanced in cells from human patients with Niemann Pick type A, a disease caused by a deficiency of ASM activity resulting in sphingomyelin accumulation. Furthermore, the addition of sphingomyelin to cultured cells also increased WNV infection whereas treatment with pharmacological inhibitors of sphingomyelin synthesis reduced WNV infection. Confocal microscopy analyses confirmed the association of SM with viral replication sites within infected cells. Our results unveil that sphingomyelin metabolism regulates flavivirus infection in vivo and propose sphingomyelin as a suitable target for antiviral design against WNV.