Extracellular vesicles (EVs), especially exosomes, are cell-derived nanoparticles harboring various cellular components such as RNAs, lipids, and proteins for intercellular communication. Roles of EVs as intercellular communicators have been extensively studied in the last few decades, especially under various pathological conditions. Deciphering the message in EVs isolated from biological fluids of patients can provide valuable information not only for disease diagnosis, but also for disease monitoring or treatment responses. EVs are also attractive treatment modality and drug delivery system with favorable properties of biocompatibility, selective tropism, and stability. Stem cell-derived naïve EVs have been tested for their regenerative or immunomodulatory effects in numerous preclinical and clinical studies. This so-called “cell-free cell therapy” is supported by the idea that most therapeutic actions of conventional cell therapy are mediated by paracrine action of EVs released from stem cells. In that sense, immune cell-derived EVs are regarded as a reasonable option for cancer immunotherapy. Such therapeutic effect of EVs can be dramatically augmented by incorporating active pharmaceutical ingredients (APIs) to make engineered exosomes as “Trojan Horses”. Biomimetic EVs or cell-derived nanovesicles can be generated through various physicochemical methos such as serial extrusion. They provide alternative options due to their high productivity and relatively easy purification. In this special issue, therapeutic applications of naïve or engineered EVs are discussed in various human diseases including cardiovascular diseases, renal disorders, neurological diseases, cancers, and infectious diseases focusing on COVID-19.