Herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope glycoprotein B (gB) in infected cells. This phosphorylation downregulates cell surface expression of gB and plays a role in viral pathogenesis in the mouse herpes stromal keratitis model. In the present study, we demonstrated that Us3 phosphorylation of gB Thr-887 upregulated the accumulation of endocytosed gB from the surfaces of infected cells. We also showed that two motifs in the cytoplasmic tail of gB, tyrosine at position 889 (Tyr-889) and dileucines at positions 871 and 872, were required for efficient downregulation of gB cell surface expression and upregulation of accumulation of endocytosed gB in infected cells. A systematic analysis of mutations in these three sequences in gB suggested that the expression of gB on the surfaces of infected cells was downregulated in part by the increase in the accumulation of endocytosed gB, which was coordinately and tightly regulated by the three gB trafficking signals. Tyr-889 appeared to be of predominant importance in regulating the intracellular transport of gB and was linked to HSV-1 neurovirulence in mice following intracerebral infection. These observations support the hypothesis that HSV-1 evolved the three gB sequences for proper regulation of gB intracellular transport and that this regulation plays a critical role in diverse aspects of HSV-1 pathogenesis.