Recent studies have revealed that diabetes mellitus caused gut bacterial dysbiosis, and intervention studies aiming to selectively alter the composition and metabolism of the gut microbiota are crucial next steps for investigating the links between the microbiome and diabetes. The sequences encompassing V1–3 16S rDNA hypervariable regions were PCR amplified from rat fecal samples fed with resistant starch (RS). In total, 13 different phyla and 107 different genus were obtained with a 3% distance cut off. The microbiota profile of normal rats was significantly different from that of rats in diabetic control and RS intervention group. The most prominent phyla were Firmicutes and Bacteroidetes in the three groups. The proportion of Proteobacteria was significantly higher in the rats of diabetic control compared to normal group, and RS intervention could inhibit the proliferation of Proteobacteria phyla, including a wide variety of pathogenic species, such as Escherichia-Shige, Klebsiella, and Pseudomona. At genus level, Lactobacillus in the diabetic control was significantly lower than that in the normal group, whereas the feeding of RS increased the amount of Lactobacillus. More importantly, the consumption of RS reduced the composition of Ruminococcus and increased S24-7 norank comparing with the diabetic control. This study might indicate that RS is an effective food ingredient for manipulating the gut microbiota.