Abstract MicroRNAs (miRNAs) play a crucial role in regulating plant growth, development and responses to biotic and abiotic stresses. Our previous studies showed that tobacco curly shoot virus (TbCSV) causes miRNA differential expression in infected Nicotiana benthamiana , but the relationship between TbCSV infection and miRNA is unclear. TbCSV infection leads to a decrease in the expression levels of nbe‐miR403‐5p. This study aimed to explore the relationship between nbe‐miR403‐5p expression and TbCSV DNA accumulation by manipulating its expression levels using pCVA for upregulation and PVX for downregulation. The results showed that increased expression of nbe‐miR403‐5p exacerbated leaf curling symptoms and enhanced viral DNA accumulation in N. benthamiana , whereas decreased expression mitigated viral DNA accumulation and alleviated symptoms of infection. Downregulation of NbAGO2 , the target gene of nbe‐miR403‐5p, consequent to the overexpression of nbe‐miR403‐5p, was identified. Silencing of NbAGO2 through virus‐induced gene silencing (VIGS) compromised plant resistance to viral infection and facilitated virus accumulation in N. benthamiana . This study sheds light on the role of nbe‐miR403‐5p in plant defence against TbCSV infection and provides insights into the molecular mechanisms underlying miRNA‐mediated interactions between plants and viruses.