Abstract: To explore the molecular mechanisms underlying Nicotiana benthamiana responses to Potato Virus Y (PVY) infection, small RNA sequencing (sRNA-seq) and transcriptome sequencing (RNA-seq) were employed to analyze the differences in tsRNAs and transcriptional responses in N. benthamiana following PVY infection. The results showed that 39 differentially expressed tiRNAs (tRNA halves) and 119 tRFs (tRNA-related fragments) were identified at the 7th day post-inoculation. Enrichment analysis of differentially expressed target genes (DETGs) demonstrated that 117 and 602 target genes of tRFs in leaves and stems, respectively, were predominantly enriched in pathways such as signal transduction and energy metabolism. Notably, the expression of tDR-1:18-His-GTG-1-M3 was most significantly downregulated in PVY-infected leaves. Suppressing the expression of tDR-1:18-His-GTG-1-M3 in N. benthamiana markedly increasing the expression levels of its target genes, yabD and RNF170. These findings provide a reference for further in-depth research on the role of tsRNAs in PVY-host plant interactions and the development of PVY control strategies.