The final step in the egress of herpes simplex virus (HSV) virions requires virion-laden vesicles to bypass cortical actin and fuse with the plasma membrane, releasing virions into the extracellular space. Little is known about the host or viral proteins involved. In the current study, we noted that the conformation of myosin Va (myoVa), a protein known to be involved in melanosome and secretory granule trafficking to the plasma membrane in melanocytes and neuroendocrine cells, respectively, was altered by 4 h after infection with HSV-1 such that an N-terminal epitope expected to be masked in its inactive state was rendered immunoreactive. Wild-type myoVa localized throughout the cytoplasm and to a limited extent in the nuclei of HSV-infected cells. Two different dominant negative myoVa molecules containing cargo-binding domains but lacking the lever arms and actin-binding domains colocalized with markers of the trans-Golgi network (TGN). Expression of dominant negative myoVa isoforms reduced secretion of HSV-1 infectivity into the medium by 50 to 75%, reduced surface expression of glycoproteins B, M, and D, and increased intracellular virus infectivity to levels consistent with increased retention of virions in the cytoplasm. These data suggest that myoVa is activated during HSV-1 infection to help transport virion- and glycoprotein-laden vesicles from the TGN, through the cortical actin, to the plasma membrane. We cannot exclude a role for myoVa in promoting fusion of these vesicles with the inner surface of the plasma membrane. These data also indicate that myoVa is involved in exocytosis in human epithelial cells as well as other cell types.