miRNA participates in various physiological and biochemical processes in plants by regulating corresponding target genes. NAC transcription factors, usually as the targets of miR164, play important roles in regulation of plant development and responses to abiotic and biotic stresses. In a previous study, the target gene of tae-miR164 in wheat was sequenced through degradome sequencing. In this study, we isolated the full-length cDNA of the candidate target gene that is a NAC transcription factor gene in the NAM subfamily, and designated it as TaNAC21/22 after bioinformatics analysis. The interaction between TaNAC21/22 and tae-miR164 was confirmed experimentally through co-transformation of both genes in tobacco leaves. Transcript accumulation of TaNAC21/22 and tae-miR164 showed contrasting divergent expression patterns in wheat response to Puccinia striiformis f. sp. tritici (Pst). TaNAC21/22 was confirmed to be located in the nucleus and could function as a transcriptional activator. Silencing of individual gene showed that TaNAC21/22 negatively regulates resistance to stripe rust. These results indicate that the target of tae-miR164, a novel NAC transcription factor from the NAM subfamily of wheat, plays an important role in regulating resistance of host plants to stripe rust.