Progressive multifocal leukoencephalopathy is a crimpling demyelinating disease of the central nervous system caused by JC polyomavirus (JCPyV). Much about JCPyV propagation in the brain remains obscure because of a lack of proper animal models to study the virus in the context of the disease, thus hampering efforts toward the development of new antiviral strategies. Here, having established a robust and representative model of JCPyV infection in human-induced pluripotent stem cell-derived astrocytes, we are able to fully characterize the effect of JCPyV on the biology of the cells and show that the proteomic signature observed for JCPyV-infected astrocytes is extended to extracellular vesicles (EVs). These data suggest that astrocyte-derived EVs found in body fluids might serve as a rich source of information relevant to JCPyV infection in the brain, opening avenues toward better understanding the pathogenesis of the virus and, ultimately, the identification of new antiviral targets.