G-quadruplex (G4) is a unique secondary structure formed by guanine-rich nucleic acid sequences. Growing studies reported that the genomes of some viruses harbor G4 structures associated with viral replication, opening up a new field to dissect viral infection. Porcine reproductive and respiratory syndrome virus (PRRSV), a representative member of Arteriviridae, is an economically significant pathogen that has devastated the swine industry worldwide for over 30 years. In this study, we identified a highly conserved G-rich sequence with parallel-type G4 structure (named PRRSV-G4) in the negative strand genome RNA of PRRSV. Pyridostatin (PDS), a well-known G4-binding ligand, stabilized the PRRSV-G4 structure and inhibited viral replication. By screening the proteins interacting with PRRSV-G4 in PRRSV-infected cells and single-molecule magnetic tweezers analysis, we found that two helicases, host DDX18 and viral nsp10, interact with and efficiently unwound the PRRSV-G4 structure, thereby facilitating viral replication. Using a PRRSV reverse genetics system, we confirmed that recombinant PRRSV with a G4-disruptive mutation exhibited resistance to PDS treatment, thereby displaying higher replication than wild-type PRRSV. Collectively, these results demonstrate that the PRRSV-G4 structure plays a crucial regulatory role in viral replication, and targeting this structure represents a promising strategy for antiviral therapies.