Gliadins are a major component of wheat seed proteins. However, the complex homoeologous Gli‐2 loci (Gli‐A2, ‐B2 and ‐D2) that encode the α‐gliadins in commercial wheat are still poorly understood. Here we analyzed the Gli‐D2 locus of Xiaoyan 81 (Xy81), a winter wheat cultivar. A total of 421.091 kb of the Gli‐D2 sequence was assembled from sequencing multiple bacterial artificial clones, and 10 α‐gliadin genes were annotated. Comparative genomic analysis showed that Xy81 carried only eight of the α‐gliadin genes of the D genome donor Aegilops tauschii, with two of them each experiencing a tandem duplication. A mutant line lacking Gli‐D2 (DLGliD2) consistently exhibited better breadmaking quality and dough functionalities than its progenitor Xy81, but without penalties in other agronomic traits. It also had an elevated lysine content in the grains. Transcriptome analysis verified the lack of Gli‐D2 α‐gliadin gene expression in DLGliD2. Furthermore, the transcript and protein levels of protein disulfide isomerase were both upregulated in DLGliD2 grains. Consistent with this finding, DLGliD2 had increased disulfide content in the flour. Our work sheds light on the structure and function of Gli‐D2 in commercial wheat, and suggests that the removal of Gli‐D2 and the gliadins specified by it is likely to be useful for simultaneously enhancing the end‐use and health‐related traits of common wheat. Because gliadins and homologous proteins are widely present in grass species, the strategy and information reported here may be broadly useful for improving the quality traits of diverse cereal crops.