Porcine reproductive and respiratory syndrome virus (PRRSV) has become one of the most economically important diseases to the global pig industry. RNase L is a ubiquitous cellular endoribonuclease that is activated upon the binding of a specific ligand, 2',5'-linked oligoadenylates (2-5 A), which is synthesized by oligoadenylate synthetases (OASs). However, whether Sus scrofa RNase L (sRNase L) could inhibit PRRSV replication and its mechanism have not been fully elucidated. In this study, sRNase L was cloned and characterized in homology and structure firstly. Then the antiviral activity of sRNase L against PRRSV was explored. Overexpression of sRNase L significantly inhibited the propagation of PRRSV when treated with 2-5 A or poly(I: C) or mock treated. Furthermore, sRNase L induced degradation of cellular and viral ssRNAs, enhanced the activation of IFN-β promoter and IFN-β expression, and induced apoptosis to inhibit PRRSV replication. Taken together, we have first elucidated the anti-PRRSV function and the underlying mechanism of sRNase L, which may provide a new strategy for preventing PRRSV infection.