Previous studies have indicated that inhibition of type I interferon production may be an important reason for porcine reproductive and respiratory syndrome virus (PRRSV) to achieve immune escape, revealing the mechanism of inhibiting the production of type I interferon will help design novel strategies for controlling PRRS. Here, we found that PRRSV infection upregulated the expression of miR-382-5p, which in turn inhibited polyI:C-induced the production of type I interferon by targeting heat shock protein 60 (HSP60), thus facilitating PRRSV replication in MARC-145 cells. Furthermore, we found that HSP60 could interact with mitochondrial antiviral signaling protein (MAVS), an important signal transduction protein for inducing production of type I interferon, and promote polyI:C-mediated the production of type I interferon in a MAVS-dependent manner. Finally, we also found that HSP60 could inhibit PRRSV replication in a MAVS-dependent manner, which indicated that HSP60 was a novel antiviral protein against PRRSV replication. In conclusion, the study demonstrated that miR-382-5p was upregulated during PRRSV infection and may promote PRRSV replication by negatively regulating the production of type I interferon, which also indicated that miR-382-5p and HSP60 might be the potential therapeutic targets for anti-PRRSV.,? 2020 Federation of American Societies for Experimental Biology.