Abstract: Opuntia ficus-indica is rich in a variety of active substances, and the anthocyanins contained in it play an important role in regulating gut microbiota. In this paper, the simulated digestion and in vitro anaerobic fermentation of Opuntia ficus-indica anthocyanins were studied in K group (blank), anthocyanin H (high dose 15 mg/mL), M group (medium dose 10 mg/mL), L group (low dose 5 mg/mL) on human gut microbes and metabolites short-chain fatty acids (SCFAs). The results showed that, using the pH test and difference method, and taking the digestibility as an indicator, the anthocyanins of Opuntia ficus-indica (10 mg/mL) were obtained, the digestibility was 11.4% after 3 h of gastric digestion, 23.5% after 4 h of intestinal digestion, the remaining 65.1% of anthocyanins were not digested by the gastrointestinal tract. Using high-throughput sequencing method, through α diversity and β diversity analysis, compared with K group, anthocyanin H, M, L group could significantly improve the diversity of gut microbiota (P<0.05). In terms of microbiota composition, based on phylum-level analysis, compared with blank group, the relative abundance of Proteobacteria in each anthocyanin dose group significantly decreased (P<0.05). The ratio of phylum Firmicutes/Bacteroidetes (F/B value) was significantly reduced (P<0.05). At the genus level, compared with the K group, the H, M and L dose groups could significantly reduce the relative abundance of Escherichia-Shigella and Desulfovibrio pathogens (P<0.05), and could significantly increase the beneficial effects. The abundance of bacteria, among which, the abundance of Prevotella in the middle-dose group was the highest, reaching 41.8%. The abundance of Lactobacillus and Bifidobacterium in the high-dose group was the highest, reaching 7.9% and 5.9%, respectively. The relative abundance of Mitsuokella in the low-dose group was greatly increased, reaching 37.7%. The changes of short-chain fatty acids were analyzed by gas chromatography (GC). Compared with the K group, the total short-chain fatty acid content of the high, medium and low dose groups significantly increased (P<0.05), and were 7.8, 5.0 and 1.1 times that of the blank group, respectively. Among them, the contents of acetic acid, propionic acid and butyric acid increased most significantly (P<0.05), the highest increased in acetic acid was 10.0 times that of the K group, 4.6 times for propionic acid and 10.7 times for butyric acid. In summary, Opuntia ficus-indica anthocyanins were digested in a small amount in the stomach, and a large amount was digested in the small intestine, and most of them were left. They could change the diversity and composition of microbes in the intestinal tract, and promote the production of short-chain fatty acids. This study would establish the foundation for the regulatory role of the microbiota and the development of functional foods.