ABSTRACTUndigested amylopectin fermentation in the hindguts of humans and pigs with low digestive capacity has been proven to be a low-efficiency method of energy supply. In this study, we researched the effects and mechanisms of amylopectin fermentation on hindgut microbiota and metabolite production using anin vitrofermentation trial and ileal infusion pigs model. In addition, we also researched the effects of interaction between amylopectin and cellulose during hindgut fermentation in this study. Our results showed that amylopectin had higher short-chain fatty acid (SCFA) production and dry matter digestibility (DMD) than cellulose but was not significantly different from a mixture of amylopectin and cellulose (Amycel vitro) duringin vitrofermentation. The Amycel vitro group even had the highest reducing sugar content and amylase activity among all groups. The ileal infusion trial produced similar results to vitro fermentation trial: the mixture of amylopectin and cellulose infusion (Amycel vivo) significantly increased the levels of reducing sugar, acetate, and butyrate in the hindgut compared with the amylopectin infusion (Amy vivo). The mixture of amylopectin and cellulose infusion also resulted in increased Shannon index and probiotic colonization in the hindgut. The relative abundance ofRomboutsiain the Amycel vivo group, which was considered a noxious bacteria in the Amycel vivo group, was also significantly lower than that in the Amy vivo group. In summary, the high level of amylopectin fermentation in the hindgut was harmful to intestinal microbiota, but amylopectin partially substituted with cellulose was beneficial to SCFA production and probiotic colonization.IMPORTANCEA high-starch (mainly amylopectin) diet is usually accompanied by the fermentation of undigested amylopectin in the hindgut of humans and pigs with low digestive capacity and might be detrimental to the intestinal microbiota. In this research, we investigated the fermentation characteristics of amylopectin through anin vitrofermentation method and used an ileal infusion pig model to verify the fermentation trial results and explore the microbiota regulatory effect. The interaction effects between amylopectin and cellulose during hindgut fermentation were also researched in this study. Our research revealed that the large amount of amylopectin fermentation in the hindgut was detrimental to the intestinal microbiota. Amylopectin partially substituted by cellulose was not only beneficial to antioxidant ability and fermentation efficiency, but also promoted SCFA production and probiotic colonization in the hindgut. These findings provide new strategies to prevent intestinal microbiota dysbiosis caused by amylopectin fermentation.