Abstract: Starch was the main source of carbohydrates in people's daily diet, and its digestibility was an important factor affecting the body's postprandial blood glucose level. In this paper, the effect of xylose on the in vitro digestive properties of corn starch and its mechanism of action were investigated. The in vitro digestibility of starch was evaluated by an in vitro simulated digestion system after different additions of xylose (5%, 10%, and 15%), and the mechanisms of xylose on α-amylase and α-glucosidase, as well as its effect on blood glucose fluctuation through enzyme inhibition kinetics, molecular docking, and human glycemic index (GI) tests. The results showed that the digestive hydrolysis of corn starch was inhibited by different amounts of xylose, and the IC₅₀ values of xylose on α-glucosidase and α-amylase were 205.35 and 4.75 mg/mL, respectively, which belonged to the mixed type inhibitors. Xylose interacted with amino acid residues of α-glucosidase and α-amylase through hydrogen bonding with binding energies of −3.05 and −5.30 kcal/mol, respectively. Additionally, the GI value of plant-based peptidoglycan milk decreased from 58 to 45 after the addition of xylose, which demonstrated that xylose was effective in decreasing the fluctuation of postprandial blood glucose. The results could provide scientific data to support the research and application of xylose in nutritional and health foods.