Xylitol is a widely used natural sweetener for the reduction of excessive sugar consumption. However, concerns of xylitol consumption existed as it is a highly permeable substance in the colon that could cause diarrhea and other adverse symptoms. To assess the relationship between xylitol dosage and diarrhea, especially the influences of diarrhea on physiological characteristics, the immune system, and gut microbiota in rats, the control, low-dose (L), medium-dose (M), and high-dose (H) groups were fed with 0, 1, 3, and 10% of xylitol, respectively, correspondingly for 15 days, followed by a 7-day recovery. Only medium- and high-dose xylitol would cause diarrhea in rats. Quantitative imaging of colonic tissue and the expression levels of proinflammatory factors revealed a higher degree of immune responses in the rats from H groups but statistically stable in M groups, despite that light diarrhea was observed. A shift of the gut microbiota composition was observed in the rats from H groups, including significant decreases of genera Ruminococcaceae and Prevotella and a notable increase and colonization of Bacteroides, accompanied with changes of short-chain fatty acid production. Tolerance and adaptation to xylitol consumption were observed in a dose-dependent manner. Our findings demonstrate that diarrhea caused by the high dosage of xylitol can exert distinctive changes on gut microbiota and lay the foundation to explore the mechanism underlying the shift in gut microbiota composition.