Porcine reproductive and respiratory syndrome virus (PRRSV) is a variable virus, whose spread cannot be totally stopped by vaccination. PRRSV infection results in abortion and respiratory symptoms in pregnant pigs. One crucial component of the anti-viral infection strategy is microRNA (miRNA), a class of multifunctional small molecules. It is unknown whether miR-339-5p can specifically target the PRRSV gene and prevent the virus from replicating, despite the fact that miR-339-5p is markedly up-regulated during the PRRSV infection. In this pursuit, the present study revealed that the two PRRSV areas targeted by miR-339-5p were PRRSV nsp2-3378 to 3403 and PRRSV nsp2-3112 to 3133 using the miRanda program. Dual luciferase reporter assays showed that the miR-339-5p target region of the PRRSV gene sequence exhibited 100% homology and was highly conserved. Furthermore, the ability of miR-339-5p to target PRRSV gene areas was verified. It was found that the overexpression of miR-339-5p markedly reduced the PRRSV replication through PRRSV infection trials. The precursor sequence of ssc-miR-339-5p was amplified using the DNA of pig lung tissue as a template in order to create a fragment of 402 bp of porcine-derived miR-339-5p precursor sequence, which was then used to produce the eukaryotic expression plasmid of miR-339-5p. In conclusion, miR-339-5p can target the specific PRRSV gene areas and prevent PRRSV replication, offering fresh perspectives for the creation of medications that combat the PRRSV infection.© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.