Coxsackievirus (CVB) is a relatively common enterovirus belonging to the picornaviridae family that is capable of causing aseptic meningitis, pancreatitis and myocarditis in humans. Coxsackievirus B3 (CVB3) preferentially targets neural progenitor and stem cells (NPSCs) with lasting consequences in the central nervous system (CNS). We genetically engineered a recombinant coxsackievirus expressing "fluorescent timer" protein (Timer-CVB3)."Fluorescent timer" protein undergoes a slow conversion from green to red over time, and thus, Timer-CVB3 can be utilized to detect the progression of virus infection. Upon infection with Timer-CVB3, partially differentiated neural progenitor and stem cells (NPSCs) and C2C12 myoblast cells released extracellular microvesicles (EMVs) containing matured "fluorescent timer" protein and infectious virus. Purified EMVs isolated from the supernatants of Timer-CVB3 infected cells were positive for the exosomal marker - flotillin- 1, the autophagosomal marker - LC3-II, and viral capsid protein - VP-1. EMVs isolated by iodixanol isopycnic density purification were found in low density fractions consistent with membrane association distinct from high density viral fractions representing infectious virions. Detection of the lipidated form of LC3 protein (LC3-II) in EMVs observed within infected cell culture supernatants suggests involvement of the autophagy pathway during the release of EMVs. Also, viral protein co-localized with LC3 protein within EMVs identified in infected cultured cells, indicating that autophagy is an important process involved in release of shed microvesicles harboring infectious CVB3. Clarifying the role of these infectious EMVs is important for understanding virus immune evasion and for the development of new antiviral therapies and vaccines.