For a variety of viruses, the primary virus infection has been shown to prevent superinfection with a homologous secondary virus; however, the mechanism of exclusion has not been clearly understood. In this work, we demonstrated that BVDV -infected MDBK cells were protected from superinfection with a homologous superinfecting BVDV, one of the positive-sense RNA pestiviruses, but not with an unrelated rhabdovirus, such as vesicular stomatitis virus. Once superinfection exclusion was established by a primary infection with BVDV, the transfected infectious BVD viral RNA genome was shown to be competent for viral translation, but not viral replication. In addition, our results also demonstrated that upon superinfection, the. viral RNA genome of viral particles was not transferred into the cytoplasm of BVDV -infected cells. Using newly developed system involving rapid generation of the MDBK cells expressing BVD viral proteins, we subsequently found that expression of the viral structural proteins was dispensable for the block occurring at the level of viral RNA replication, but required for the exclusion at the level of viral entry step. Altogether, these findings provide evidence that the superinfection exclusion of BVDV occurs not only at the level of viral replication in which the viral replicase are involved, but also at the level of viral entry with which the viral structural proteins are associated, and that a cellular factor(s) play an essential role in this process.