This study aimed to investigate the effects of water-unextractable arabinoxylan (WUAX) on the gluten network structure, especially on gliadins and glutenins. The results indicated that the free sulfhydryl (free SH) of gliadins increased by 25.5% with 100 g/kg WUAX, whereas that of glutenins increased by 65.2%, which inhibited the formation of covalent bonds. Furthermore, β-sheets content decreased 5.63% and 4.75% for gliadins and glutenins with 100 g/kg WUAX, respectively, compared with the control. WUAX increased β-turns prevalence for gliadins, while the content of α-helixes and random coils had less fluctuation. In glutenins, the contents of α-helixes and β-sheets decreased and β-turns increased. Moreover, compared with the control, the weight loss rate for gliadins and glutenins increased by 2.49% and 2.04%, respectively, with 60 g/kg WUAX. The dynamic rheological analysis manifested that WUAX impaired the viscoelasticity property of gliadin and glutenin. Overall, WUAX weakened the structure of the gliadins and glutenins, leading to quality deterioration of gluten.