Cyclin-dependent kinase 9 (CDK9) is a key regulator of RNA-polymerase II and a candidate therapeutic target for various virus infections such as respiratory syncytial virus, herpes simplex virus, human adenovirus, human cytomegalovirus, hepatitis virus B, and human papillomavirus. We employed CDK9-IN-1, a selective CDK9 inhibitor, to investigate the role of CDK9 in porcine reproductive and respiratory syndrome virus (PRRSV) infection. CDK9-IN-1 dose-dependently reduced PRRSV replication without cytotoxicity in the infected cells. The antiviral activity of CDK9-IN-1 was further confirmed by evaluating the effects of lentivirus-mediated CDK9 knockdown or CDK9 overexpression on PRRSV infection. Briefly, the depletion of CDK9 significantly inhibited viral replication, while the overexpression of CDK9 promoted viral replication. PRRSV infection also enhanced the nuclear export of CDK9 without affecting CDK9 protein expression. Viral replication cycle analyses further revealed that functionally active CDK9 in the cytosol advanced viral subgenomic RNA synthesis. Collectively, our data illustrated that CDK9 was a new host factor that was involved in PRRSV subgenomic RNA synthesis, and CDK9 inhibitor, CDK9-IN-1 was a promising antiviral candidate for PRRSV infection.