Background <p>Bovine viral diarrhoea (BVD), a condition triggered by bovine viral diarrhoea virus (BVDV), is recognized globally as a prevalent pathogen among ruminants and markedly affects the economics of animal husbandry. MicroRNAs, a class of small noncoding RNAs, play pivotal roles in regulating a myriad of biological processes.The ATG7-LC3 pathway, a canonical autophagy mechanism, is integral in defending against pathogenic invasion and maintaining cellular homeostasis.</p> Results <p>In this study, we observed significant downregulation of bta-miR-221 in cells infected with BVDV. We further established that overexpression of bta-miR-221 markedly attenuated BVDV replication in Madin‒Darby bovine kidney (MDBK) cells. Through bioinformatics prediction analysis, we identified ATG7, an autophagy-related gene, as a direct downstream target of bta-miR-221. However, the intricate relationships among bta-miR-221, the ATG7-LC3 pathway, and BVDV infection remained unclear. Our study revealed that ATG7 expression was significantly elevated in BVDV-infected cells, whereas bta-miR-221 mimics repressed both endogenous and exogenous ATG7 expression. Following BVDV infection, we noted a decrease in LC3I expression, its conversion to LC3II, a significant increase in ATG7 expression, and a notable decrease in SQSTM1/p62 expression. By employing laser confocal microscopy and immunoprecipitation assays, we elucidated the regulation of the ATG7-LC3 pathway by bta-miR-221 in MDBK cells. Our findings recealed that BVDV infection enhanced the ATG7-LC3 interaction, inducing autophagy through the suppression of bta-miR-221 in MDBK cells. Consequently, bta-miR-221 emerged as a potent inhibitor of BVDV, impacting its proliferation and replication within the host.</p> Conclusions <p>This research sheds light on novel aspects of virus-host interactions and lays a foundation for the development of antiviral therapeutics.</p>