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As an alternative to traditional dietary fibers with prebiotic effects, the interaction between resistant starch III (RS3) and gut microbiota is worth exploring. In this study, the effects of RS3 on the proliferation of Bifidobacterium adolescentis (B. adolescentis) and their structural changes before and after fermentation were investigated. Autoclaved-debranched resistant starch (ADRS) demonstrated the best proliferative effect for B. adolescentis and the highest roughness (Ra = 21.90 nm; Rq = 16.00 nm). The rough surface of ADRS was the key for B. adolescentis proliferation. B. adolescentis produced an extracellular amylase to assist degradation and showed the highest activity in ADRS. Fermentation disrupted short-range ordered structure and reduced R 1047 cm -1 /1022 cm -1 by 20.74 % and R 995 cm -1 /1022 cm -1 by 30.85 %. The extracellular amylase was essential substance for ADRS degradation. These findings help optimize RS3 structure and promote the proliferation of intestinal probiotics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)