In Arabidopsis, NPR1 is a key transcriptional coregulator of systemic acquired resistance. Upon pathogen challenge, NPR1 translocates from the cytoplasm to the nucleus, in which it interacts with TGA-bZIP transcription factors to activate the expression of several pathogenesis-related (PR) genes. In a screen of a yeast two-hybrid library from wheat leaves infected with Puccinia striiformis f. sp. tritici, we identified a conserved rust protein that interacts with wheat NPR1 and named it PNPi (for Puccinia NPR1 interactor). PNPi interacts with the NPR1/NIM1-like domain of NPR1 via its C-terminal DPBB_1 domain. Using bimolecular fluorescence complementation assays, we detected the interaction between PNPi and wheat NPR1 in the nucleus of Nicotiana benthamiana protoplasts. A yeast three-hybrid assay showed that PNPi interaction with NPR1 competes with the interaction between wheat NPR1 and TGA2.2. In barley transgenic lines overexpressing PNPi, we observed reduced induction of multiple PR genes in the region adjacent to Pseudomonas syringae pv. tomato DC3000 infection. Based on these results, we hypothesize that PNPi has a role in manipulating wheat defense response via its interactions with NPR1.