The human cytomegalovirus tegument protein UL69 has been shown to be required for efficient viral replication at low multiplicities of infection. Several functions have been associated with UL69, including its ability to regulate cell cycle progression, translation, and the export of viral transcripts from the nucleus to the cytoplasm. However, it remains unclear which, if any, of these activities contribute to the phenotype observed with the UL69 deletion mutant. UL69 has been shown to interact with the cellular protein SPT6. The functional significance of this interaction has never been examined in the context of an infection. To address this, we generated UL69 mutant viruses that were unable to interact with SPT6 and determined what effect these mutations had on virus replication. Abolishing UL69's ability to interact with the SPT6 protein inhibited virus replication to levels indistinguishable from those observed following infection with the UL69 deletion mutant. Surprisingly, abolishing UL69's interaction with SPT6 also resulted in the impairment of UL69 shuttling activity. Finally, we demonstrate that inhibition of SPT6 expression by short hairpin RNA (shRNA) knockdown inhibits wild-type virus replication. Taken together, our results demonstrate that UL69's ability to interact with SPT6 plays a critical role in viral replication.