Viruses remodel the host cell to optimize their replication both by delivery of virion proteins into the cell and by de novo expression of viral proteins. The HSV particle contains several proteins that function to prepare the host cell for viral replication, including the VP16 transcriptional activator protein and virion host shutoff protein. HSV infection activates NF-kappaB pathways through Toll-like receptor (TLR) 2 and non-TLR pathways, and NF-kappaB activity is required for efficient viral replication. In a screen of the HSV proteome, we observed that the HSV U(L)37 tegument protein activates NF-kappaB signaling in a TLR2-independent manner. Expression of U(L)37 in transfected cells leads to IkappaB degradation and activation of both reporter genes and the endogenous IL-8 gene. This activation requires TNF receptor-associated factor 6 (TRAF6), and U(L)37 contains a TRAF6-binding domain that is required for interaction with TRAF6 and activation of NF-kappaB. A mutant virus encoding U(L)37 with an altered TRAF6-binding site shows reduced NF-kappaB activation in the early phase of infection. Therefore, the HSV U(L)37 virion structural protein can activate NF-kappaB through TRAF6. Activation of NF-kappaB by a virion tegument protein that is delivered into the host cell cytoplasm during viral entry represents a mechanism for activation of this pathway by a virus.