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Effectors are considered to be virulence factors secreted by pathogens, which play an important role during host-pathogen interactions. In this study, the candidate effector Pt9226 was cloned from genomic DNA of <i>Puccinia triticina</i> (<i>Pt</i>) pathotype THTT, and there were six exons and five introns in the 877 bp sequence, with the corresponding open reading frame of 447 bp in length, encoding a protein of 148 amino acids. There was only one polymorphic locus of I142V among the six <i>Pt</i> pathotypes analyzed. Bioinformatics analysis showed that Pt9226 had 96.46% homology with the hypothetical putative protein PTTG_26361 (OAV96349.1) in the <i>Pt</i> pathotype BBBD. RT-qPCR analyses showed that the expression of Pt9226 was induced after Pt inoculation, with a peak at 36 hpi, which was 20 times higher than the initial expression at 0 hpi, and another high expression was observed at 96 hpi. No secretory function was detected for the Pt9226-predicted signal peptide. The subcellular localization of Pt9226^Δsp-GFP was found to be multiple, localized in the tobacco leaves. Pt9226 could inhibit programmed cell death (PCD) induced by BAX/INF1 in tobacco as well as DC3000-induced PCD in wheat. The transient expression of <i>Pt9226</i> in 26 wheat near-isogenic lines (NILs) by a bacterial type III secretion system of <i>Pseudomonas fluorescens</i> EtHAn suppressed callose accumulation triggered by Ethan in wheat near-isogenic lines TcLr15, TcLr25, and TcLr30, and it also suppressed the ROS accumulation in TcLr15. RT-qPCR analysis showed that the expression of genes coded for pathogenesis-related protein TaPR1, TaPR2, and thaumatin-like protein TaTLP1, were suppressed, while the expression of <i>PtEF-1α</i> was induced, with 1.6 times at 72 h post inoculation, and TaSOD was induced only at 24 and 48 h compared with the control, when the <i>Pt</i> pathotype THTT was inoculated on a transient expression of <i>Pt9226</i> in wheat TcLr15. Combining all above, Pt9226 acts as a virulence effector in the interaction between the <i>Pt</i> pathotype THTT and wheat.