Background: Yam (Dioscorea opposita Thunb.) has been consumed as a food and used in traditional Chinese medicine for thousands of years. Resistant starch (RS) 3 is of particular interest because it is heat-resistant, safe and non-toxic, and retains good nutritional benefits; it is therefore used in a wide range of traditional and emerging foods as a heat-stable prebiotic ingredient. In our previous study, we found that yam RS includes strong lipid-lowering and anti-constipation activities. Methods: Yam RS3 was prepared by autoclaving-retrogradation and pullulanase debranching to yield autoclaving-retrogradation yam RS and pullulanase debranching yam RS, respectively. First, the physicochemical properties of both RS3s were analyzed. Second, the structures of the RS3s were characterized by scanning electron microscopy, X-ray powder diffraction, and Fourier transform infrared spectroscopy. Finally, the regulatory effects of the RS3s on the gut microbiota were evaluated using an in vitro fecal fermentation model. Results: The RS content of the RS3s decreased after processing, but was higher in pullulanase debranching yam RS (35.67%) than in autoclaving-retrogradation yam RS (28.71%). Compared with native yam starch, RS3s lost their original granular shapes and instead exhibited irregularly shapes with continuous phases. The crystalline structure of the RS3s was completely altered, with pullulanase debranching yam RS exhibiting B-type patterns. Both RS3s, and especially pullulanase debranching yam RS, promoted a significant increase in short chain fatty acid content after in vitro fermentation (all P < 0.05). Moreover, pullulanase debranching yam RS significantly increased the abundance of beneficial bacteria and decreased the abundance of harmful bacteria such as Escherichia and Shigella (all P < 0.05). Conclusion: Our findings show that yam RS3s can regulate the composition of the gut microbiota and promote the production of short chain fatty acid, especially butyric acid. Pullulanase debranching was a more effective method for producing functional yam RS3.