Viruses that cause systemic disease often spread through the bloodstream to infect target tissues. Although viremia is an important step in the pathogenesis of many viruses, how viremia is established is not well understood. Reovirus has been used to dissect mechanisms of viral pathogenesis and is being evaluated in clinical trials as an oncolytic agent. After peroral entry into mice, reovirus replicates within the gastrointestinal tract and disseminates systemically via hematogenous or neural routes. Junctional adhesion molecule-A (JAM-A) is a tight junction protein that serves as a receptor for reovirus. JAM-A is required for establishment of viremia and viral spread to sites of secondary replication. JAM-A also is expressed on the surface of circulating hematopoietic cells. To determine contributions of endothelial and hematopoietic JAM-A to reovirus dissemination and pathogenesis, we generated strains of mice with altered JAM-A expression in these cell types and assessed bloodstream spread of reovirus strain type 1 Lang (T1L), which disseminates solely by hematogenous routes. We found that endothelial JAM-A but not hematopoietic JAM-A facilitates reovirus T1L bloodstream entry and egress. Understanding how viruses establish viremia may aid in development of inhibitors of this critical step in viral pathogenesis and foster engineering of improved oncolytic viral vectors.