Resistant starch is a kind of important carbon source for colonic microorganisms. The study of its structure-function relationship is helpful to understand the mechanism of dietary nutrition in the body. In this paper， lotus seed resistant starch prepared by microwave-power method (MP-LRS3) and water-bath method (WB-LRS3) were used as raw materials. With digestion in vitro， MP-LRS3 and WB-LRS3 were used to determine the structural changes and established their nutritional interactions of Bifidobacterium breve. The results showed that after digestion in vitro， the crystallinity of MP-LRS3 and WB-LRS3 changed significantly(P<0.05)， the layered structure with rough surface gradually depolymerizated into smaller particles with rounded corners， and both of their crystal structure still were B type. After recrystallization， the double helix structure was compacter and the arrangement of starch molecules was more orderly. However， due to the different preparation methods， the crystal properties were different after digestion. At the same digestion stage， the depolymerization degree of MP-LRS3 was higher than that of WB-LRS3. In addition， MP-LRS3 and WB-LRS3 showed a better adhesion reaction to Bifidobacterium breve with the process of digestion， but the level of the former was higher at the same digestion stage， which might be related to the different depolymerization degree in the digestion environment of lotus seed resistant starch caused by the structural differences. There was a positive correlation between the adhesion reaction and the ability to promote proliferation. It was speculated that the structural difference caused by the different degree of depolymerization made that MP-LRS3 was stronger than WB-LRS3 on adhesion ability and proliferative effect. The results of this study could provide a theoretical basis for improving the metabolism and probiotic action of resistant starch in vivo.