Resistant starch type 3 (RS3) benefits colon health, but the molecular structural reasons for this effect are unclear. Five rice starches with varied amylose content (19.1 %-40.6 %) were used to investigate their effect on gut microbiota. Size-exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis were used to characterize whole starch molecular size distributions and chain length distributions. It was found that RS3 with more chains of degree of polymerization (DP) 36-100 and smaller molecular size can promote the relative abundance of some classes of gut bacteria, while other classes were promoted by RS3 with fewer chains of DP 36-100 and larger molecular size. X-ray diffraction and scanning electron microscopy showed that crystallinity types B or C and differences in physical surface affected the microbiota. This study shows that RS3s with different fine structures are utilized differently by gut microbiota, which may be applied to develop functional foods for gut health.