Beet necrotic yellow vein virus (BNYVV) infections induce stunting and leaf curling, as well as root and floral developmental defects and leaf senescence in Nicotiana benthamiana . A microarray analysis with probes capable of detecting 1596 candidate microRNAs (miRNAs) was conducted to investigate differentially expressed miRNAs and their targets upon BNYVV infection of N. benthamiana plants. Eight species-specific miRNAs of N. benthamiana were identified. Comprehensive characterization of the N. benthamiana microRNA profile in response to the BNYVV infection revealed that 129 miRNAs were altered, including four species-specific miRNAs. The targets of the differentially expressed miRNAs were predicted accordingly. The expressions of miR164, 160, and 393 were up-regulated by BNYVV infection, and those of their target genes, NAC21/22 , ARF17/18 , and TIR , were down-regulated. GRF1 , which is a target of miR396, was also down-regulated. Further genetic analysis of GRF1 , by Tobacco rattle virus -induced gene silencing, assay confirmed the involvement of GRF1 in the symptom development during BNYVV infection. BNYVV infection also induced the up-regulation of miR168 and miR398. The miR398 was predicted to target umecyanin , and silencing of umecyanin could enhance plant resistance against viruses, suggesting the activation of primary defense response to BNYVV infection in N. benthamiana . These results provide a global profile of miRNA changes induced by BNYVV infection and enhance our understanding of the mechanisms underlying BNYVV pathogenesis.