This study explored the effects of glutelin digestion during rice grain storage on its biological activity and ability to modulate gut microbiota. In addition to structural changes, rice glutelin was investigated for its antioxidant activity, angiotensin-converting enzyme inhibition ability, properties of the peptides generated by its digestion, and effect on gut microbial composition. After 2 days storage, β-sheet content increased from 65.60 ± 2.77% to 71.15 ± 0.42%, fluorescence intensity reached the maximum, as well as the ACE inhibitory ability, Fe2+ chelating, ABTS+ and DPPH· scavenging, and the cellular anti-oxidation (CAA) activity of digested glutelin. However, prolonged storage for 3–7 days led to increased β-turn content and continuous decrease in biological activity. Moreover, the molecular weight of the peptides generated during glutelin digestion increased from to 700–1000 kDa to 1000–1800 kDa Chao1 and Shannon indices of gut microbiota increased during 1–3 days storage and decreased during 4–7 days. Enterococcus and Bacteroides increased, whereas Streptococcus, Lactobacillus, Faecalibacterium, Parasutterella, and Turicibacter decreased during glutelin storage. Thus, the stability of glutelin structure contributed to improved biological activity on digestion and promoted gut microbial community richness, while long-term storage weakened its biological activity and ability to regulate gut microbiota.