2-O-β-D-Glucopyranosyl-L-ascorbic acid (AA-2βG) from Lycium barbarum fruits has diverse bioactivities, yet its absorption and digestion are poorly understood. Therefore, the in vivo absorption of AA-2βG in rats was investigated in the present study. After oral administration to SD rats, AA-2βG was absorbed intact, reaching a peak plasma concentration of 472.32 ± 296.64 nM at 90 min, with fecal excretion peaking at 4-8 h and decreasing rapidly by 12-24 h, indicating a prolonged intestinal presence. Furthermore, the digestibility under simulated gastrointestinal conditions and the impact on the gut flora through in vitro fermentation of AA-2βG were investigated. The results reveal that AA-2βG resisted in in vitro simulated digestion, indicating potential interactions with the gut microbiota. The results of in vitro fermentation showed that AA-2βG regulated the composition of the gut microbiota by promoting Oscillospiraceae, Faecalibacterium, Limosilactobacillus, and Fusicatenibacter, while inhibiting Enterococcus, Phocaeicola, Bacteroides, and Streptococcus. Furthermore, at the species level, AA-2βG promoted the growth of Limosilactobacillus mucosae and Faecalibacterium prausnitzii, and inhibited the growth of Enterococcus. F. prausnitzii is a major producer of n-butyric acid, and the results of short-chain fatty acids also demonstrated a significant promotion of n-butyric acid. Therefore, the study on the absorption, excretion, and regulatory effects of AA-2βG on the gut microbiota supported its potential development as a functional food additive to enhance intestinal health and prevent diseases.