Acquisition of the membrane and genome encapsidation is an important step in the replication of enveloped viruses. The biogenesis of the poxviral primary membrane and the core as well as the mechanisms of their maturation are poorly understood. Using RNA interference approach, we demonstrate that a cellular trans-Golgi network membrane protein, golgin-97, is essential for virus replication. Analysis of the virion morphology in the cells depleted of golgin-97 shows that the protein is required for the virus morphogenesis and, in particular, for the formation of the first infectious virus form, mature virus, but not its precursor, immature virus. This suggests that golgin-97 may be involved in the maturation of the virus core and, potentially, the virus membrane.