The current study analyzed the different effects of intervention in high-fat diet fed rats using chito-oligosaccharides (CO group), resistant starch (RS group) and their complexes (CO-RS group), respectively. Properties such as fecal fat composition, gut derived fecal microbiota and fecal metabolites were investigated. The results show that supplementation with CO-RS in the high-fat diet led to the highest level of fecal fat excretion, followed by the CO and RS groups, and its fatty acid composition was characterized by a lower ∑UFA and higher ∑SFA levels. Furthermore, CO-RS consumption significantly enhanced the excretion of bile acids in the feces, which might be associated with a higher conversion of cholesterol into bile acids and the enhanced binding capacity with the bile acids. The fecal microbiota profile using amplified V4 rDNA suggested that rats in the CO-RS group developed an increased richness and diversity in the gut bacterial community compared to the high-fat diet group. More importantly, the CO-RS intake significantly increased the abundance of both Lactobacillus and Bifidobacterium, indicating their potential use as prebiotics. Furthermore, the highest abundance of Allobaculum and Blautia genus in the feces of the CO-RS group was also found in this study, which is highly related to the highest production of short-chain fatty acids (SCFAs) in the colon among all the animals groups. A histological analysis of colon tissue revealed that the CO-RS supplemented diet was associated with a greater degree of thickness of the mucosa layer compared to the rats in the untreated high-fat diet group. The resulting shift in the gut microbiome, increased metabolite (SCFAs) production and increased thickness of the mucosal layer may provide profound influences of CO-RS consumption on gut protection.