To reveal the changes in microbiota community structure and antioxidant activity of polyphenols from litchi pericarp during culture with human fecal microbiota, litchi pericarp polyphenols were added into the mixed medium of intestinal microbiota solution prepared from fresh feces of healthy volunteers for the anaerobic culture in vitro. The total phenol content and antioxidant activities were measured at 0, 4, 8, 12, 24 and 48 h. The change of intestinal microbiota was analyzed by high-throughput sequencing. The results showed that the total phenol content significantly decreased within 0~48 h and the antioxidant activity was also reduced but did not attain significant level. The value of total phenol content, FRAP and ABTS of 48 h was decreased to 79.06% (P＜0.05), 92.19% and 93.58%, respectively to that of 0 h. The abundance of families, genera and species presented an increasing trend from 0 to 48 h. Among them, the relative abundance of Fusobacterium and Desulfovibrio, as dominant bacteria increased to 13.58 and 10.95 times of that of 0 h, respectively. The relative abundance of Bacteroides and Peptostreptococcus decreased to 57.16% and 20.87%, respectively to that of 0 h. Enterococcus and Phascolarctobacterium showed a synergistic relationship, while Desulfovibrio and Bifidobacterium competed with each other. Our results showed that litchi pericarp polyphenols could change the structure of intestinal microbiota, which might play an important role in the biological activity of litchi pericarp polyphenols.