Usutu virus (USUV), an African mosquito-borne flavivirus closely related to West Nile virus, was first isolated in South Africa in 1959. USUV emerged in Europe two decades ago, causing notably massive mortality in Eurasian blackbirds. USUV is attracting increasing attention due to its potential for emergence and its rapid spread in Europe in recent years. Although mainly asymptomatic or responsible for mild clinical signs, USUV was recently described as being associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting the potential health threat posed by the virus. Despite this, USUV pathogenesis remains largely unexplored. The aim of this study was to evaluate USUV neuropathogenicity using in vivo and in vitro approaches. Our results indicate that USUV efficiently replicates in the murine central nervous system. Replication in the spinal cord and brain is associated with recruitment of inflammatory cells and the release of inflammatory molecules as well as induction of antiviral-responses without major modulation of blood-brain barrier integrity. Endothelial cells integrity is also maintained in a human model of the blood-brain barrier despite USUV replication and release of pro-inflammatory cytokines. Furthermore, USUV-inoculated mice developed major ocular defects associated with inflammation. Moreover, USUV efficiently replicates in human retinal pigment epithelium. Our results will help to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.