Wheat stripe rust, caused by Puccinia striiformis West. f. sp. tritici Eriks. ＆Henn. (Pst), is a serious fungal disease. Identification of new genes associate with stripe rust resistance is important for developing disease resistant wheat cultivars and studying the mechanism of disease resistance. Trihelix is a plant specific transcription factor family, which is involved in regulation of growth and development, morphogenesis, and response to stresses. So far, no study reports on the relationship between the Trihelix family and wheat stripe rust. In this study, a gene in the GTγ subfamily of Trihelix family, designated TuGTγ-3, was cloned from Triticum urartu Tum. (2n=2x=14, AA). The results of sequencing demonstrated that TuGTγ-3 gene consisted of a complete open reading frame (ORF), and its coding sequence was 1329 bp in length, which encoded a protein with 442 amino acids. The predicted molecular weight of this protein was 50.31 kDa and the theoretical isoelectric point was 6.12. Bioinformatic analysis revealed that TuGTγ-3 protein had a monopartite nuclear localization signal (GLPMQKKMRYT), and had neither transmembrane domain nor signal peptide. The conserved trihelix domain, the fourth α-helix and the CC domain were located in the regions of Q115?R187, F234?Y241 and K362?K436, respectively. Dissection of secondary structure showed that TuGTγ-3 protein comprised of 43.89% α-helix, 9.51% extended strand, 9.95% β-turn and 36.65% random coil structures. Based on the BLAST search against the genome database of common wheat from IWGSC, TuGTγ-3 was located on the long arm of chromosome 5A. Transient expression experiment using onion inner epidermal cell showed that the fusion protein TuGTγ-3-GFP distributed mainly in nuclear and slightly in cytoplasm. Expression profiles in different organs indicated that expression level of TuGTγ-3 was much higher in leaves than that in roots or leaf sheaths, and the expression in leaves was extremely up-regulated by infection of the Pst race CYR32. Furthermore, the BSMV-VIGS experiment demonstrated that the transcription factor TuGTγ-3 positively regulated resistance to stripe rust in T. urartu.