The blacklegged tick, Ixodes scapularis, is the most medically relevant arthropod vector of infectious disease in the United States. It transmits over six pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum, but remains critically understudied. I. scapularis releases a variety of anti-inflammatory, anesthetic, and anti-coagulant molecules through its saliva during a blood meal. Recently, it was found that extracellular vesicles (EVs) are secreted in its saliva and may impact microbial transmission to the mammalian host. EV biogenesis and function have been studied in mammalian systems, but their role in arthropods of public health relevance remains elusive. In mammals, the large Rab GTPase family is known to be closely associated with EV biogenesis and many of these proteins appear in the I. scapularis genome. We report that tick Rab27 is critical for tick EV biogenesis and infection. Through an in vivo model, we have shown that silencing Rab27 impacts tick fitness. Furthermore, the tick acquires less A. phagocytophilum after Rab27 silencing. In vitro experiments show that silencing of Rab27 causes a size increase of tick EVs which indicates that Rab27 is needed to regulate vesicle biogenesis. Overall, we have observed that Rab27 plays an important role in tick EV biogenesis and the tripartite interactions between vectors, hosts, and microbes.