Wheat stripe rust and powdery mildew are important worldwide diseases of wheat (Triticum aestivum). The wheat cultivar Xingmin318 (XM318) is resistant to both wheat stripe rust and powdery mildew, which are caused by Puccinia striiformis f. sp. tritici (Pst) and Blumeria graminis f. sp. tritici (Bgt), respectively. To explore the molecular mechanisms of wheat defenses against Pst and Bgt, quantitative proteomic analyses of XM318 inoculated with Pst and Bgt, respectively, were performed using tandem mass tags (TMT) technology. A total of 741 proteins were identified as differentially accumulated proteins (DAPs). Bioinformatics analyses indicated that some functional categories, including antioxidant activity, exhibited obvious differences between Pst and Bgt infections. Intriguingly, only 42 DAPs responded to both Pst and Bgt infections. Twelve DAPs were randomly selected for RT-qPCR analysis, and the mRNA expression levels of eleven were consistent with their protein expression. Furthermore, gene silencing using the virus-induced gene silencing system indicated that glutathione S-transferase (TaGSTU6) has an important role in resistance to Bgt but not to Pst. TaGSTU6 was shown to interact with the cystathionine beta-synthase (CBS) domain-containing protein (TaCBSX3). Knockdown of TaCBSX3 expression only reduced wheat resistance to Bgt infection. Overexpression of TaGSTU6 and TaCBSX3 in Arabidopsis (Arabidopsis thaliana) promoted plant resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Our results indicated that the TaGSTU6 interacting with TaCBSX3 only confers wheat resistance to Bgt, suggesting that wheat has different response mechanisms to Pst and Bgt stress.One-sentence summaryProteomics revealed a difference in the wheat resistance response to Pst and Bgt, and the TaGSTU6/TaCBSX3 interaction plays an important role only in wheat resistance to Bgt.