Monocyte/macrophage lineage cells are major target cells of porcine circovirus type 2 (PCV2). From tissues of field pigs suffering from postweaning multisystemic wasting syndrome (PMWS), both intracytoplasmic and intranuclear PCV2 signals, including antigens and nucleic acid, were easily detected in the monocyte/macrophage lineage cells. However, there was a high incidence of intracytoplasmic PCV2-positive signals, but lack of intranuclear signals and PCV2 replication in these cells in vitro. Concurrent infection with bacteria and activation of immune system are suggested to promote viral replication. In this study, lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA) were used to stimulate PCV2-inoculated alveolar macrophages (AMs). A decrease in intracytoplasmic but increase in intranuclear PCV2-positive signals, including antigens and nucleic acid, were detected in LPS-treated PCV2-inoculated AMs, but not in PMA-treated cells. Additionally, the replication product corresponding to PCV2 spliced major capsid protein (Cap) mRNA and a significant elevation in PCV2 titer were demonstrated in the LPS-treated PCV2-inoculated AMs. The results imply that Gram-negative bacterial co-infection in PCV2-infected pigs may be an important factor in promoting PCV2 replication and contributing, at least partially, to the full development of PMWS.