In the present study, we investigated the effects of sourdough on the staling rate of bread from the perspectives of starch retrogradation during storage and gluten depolymerization in bread dough. Three sourdough types, which were fermented using Weissella cibaria, Latilactobacillus sakei, and Fructilactobacillus sanfranciscensis strains, were added to bread dough at a 25% ratio. Compared with regular bread, sourdough bread (SDB) exhibited a lower hardness value and slower staling rate during a 7-day storage period. The relative crystallinities and enthalpies revealed that sourdough addition inhibited amylopectin recrystallization. Furthermore, sourdough-induced acid conditions promoted glutenin macropolymer depolymerization; and the dough containing sourdough (SD-D) showed a decrease in viscoelastic properties and changes on microstructure, reflecting a weakened gluten network structure. Among the three sourdough types, L. sakei-fermented sourdough exhibited the lowest staling rate; this might be owing to its higher gluten depolymerization degree, lower α-helix and intermolecular β-sheet contents, and decreased viscoelastic properties. Our study findings provide a theoretical basis for using sourdough fermentation with different strains to develop clean label bread and improve bread quality in the future.