The present study demonstrates how different potato virus Y (PVY) strains affect the miRNA balance in tobacco cv. Samsun. The two prevalent strains PVYNTN and PVYN-Wi caused severe and mild veinal necrosis (VN) respectively, and the unique PVYZ-NTN strain induced milder vein clearing (VCl) in the upper non-inoculated leaves. A single amino acid polymorphisms (SAPs) I252V and a Q412 to R412 substitution in the HC-Pro cistron of the PVYZ-NTN strain might relate to the loss of VN in tobacco. The abundance of 18 out of the 26 tested miRNAs was increased upon infection by the severe strains PVYNTN and PVYN-Wi. Expression of a group of defense related transcripts were increased accordingly. Two miRNAs, nta-miR6020a-5p and nta-miR6164a/b, which target the TIR-NBS-LRR type resistant TMV N genes involving in signal transduction, might correlate with the PVYNTN and PVYN-Wi induced VN. The down-regulated mRNAs, e.g., RAP2-7 and TOE3, PXC3, LRR-RLK, ATHB-14 and TCP4 targeted by nta-miR172, nta-miR390, nta-miR482, nta-miR166 and nta-miR319/159 respectively, were related to regulation of transcription, protein phosphorylation and cell differentiation. The observed strain-specific alteration of miRNAs and their targets are host dependent and corresponds to the symptom severity and the viral HC-Pro RNA levels.