Porcine reproductive and respiratory syndrome virus has caused hundreds of thousands of deaths in pig farms in many swine-producing areas in the world in recent years. However, at present there is no effective method to prevent and control the disease, and there is a need to develop new antiviral strategies. In this study, four recombinant adenoviruses expressing shRNAs targeting ORF1b, ORF5 and ORF7 were constructed, and it was found that they could down-regulate effectively specific gene expression and inhibit viral replication in MARC-145 cells when compared to the controls. They could also inhibit effectively PRRSV replication in porcine alveolar macrophages. The inhibition effect was dose-dependent and could be sustained for at least 96h in macrophages. In addition, PRRSV replication could be suppressed significantly by shRNA in cells infected previously or simultaneously with PRRSV. The results indicated that the shRNA-expressing rAd5 targeting to various gene regions of PRRSV might be a potential anti-PRRSV strategy.