Objective To investigate the effect of Amomum villosum compound on intestinal flora imbalance induced by chemotherapeutic drug 5-FU in rats. Methods The Spraque-Dawley(SD) rats were randomly divided into 6 groups, namely normal group, model group, positive drug group, and Amomum villosum low, medium and high dose groups. Based on the 16S rRNA sequencing of rat fecal bacteria and the analysis of i-Singer cloud platform data, the species diversity of intestinal flora and the abundance of flora at different taxonomical levels were analyzed. Results The results show that Amomum villosum compound can increase the diversity of intestinal flora in model group rats, and increase the abundance of Lactobacillaceae, et al; and decreased the abundance of Prevotellaceae, et al, and the best effect was obtained with the low dose of Amomum villosum compound. Conclusion Amomum villosum compound can increase the diversity of intestinal flora and regulate the homeostasis of intestinal flora at different classification levels to play an intestinal protective role.