Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication. EVs and viruses share several properties related to their structure and the biogenesis machinery in cells. EVs from virus-infected cells play a key role in virus spread and suppression using various loading molecules, such as viral proteins, host proteins, and microRNAs. However, it remains unclear how and why viruses regulate EV production inside host cells. The purpose of this study is to investigate the molecular mechanisms underlying EV production and their roles in Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected cells. Here, we found that KSHV induced EV production in human endothelial cells via Rab- 27b upregulation. The suppression of Rab27b expression in KSHV-infected cells enhanced cell death by increasing autophagic flux and autolysosome formation. Our results indicate that Rab27b regulates EV biogenesis to promote cell survival and persistent viral infection during KSHV infection, thereby providing novel insights into the crucial role of Rab- 27b in the KSHV life cycle.