Non-enveloped viruses, such as picornaviruses, can escape infected host cells via release in extracellular vesicles (EVs) prior to the induction of lysis. These EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating the formation and release of EV-enclosed viruses. The encephalomyocarditis virus (EMCV), belonging to the family of picornaviruses, expresses a Leader protein that targets various host cell processes. In this study, LC-MS/MS proteomic analysis was performed on EVs isolated via density gradient-based separation from HeLa cells infected with wildtype or Leader-deficient EMCV. Proteomes of these EVs were compared and proteins incorporated in EVs in a Leader-dependent manner were subjected to functional enrichment analysis and protein-interaction analysis.