Annealing effects on the structure characteristics and the digestibility of corn starch (CS)/corn oil (oil)/lysine mixture were investigated. The objective of this study was to provide guidance for designing higher slowly digestible starch. Confocal laser confirmed that lysine adhered to granules surface. The interactions among starch, corn oil and lysine were further investigated by using X-ray diffraction (XRD), differential scanning calorimetry (DSC) and rapid visco analyzer (RVA). After annealing treatment, in vitro digestion studies showed that the content of slowly digestible starch increased in the mixture blended with corn oil and lysine. The physical barrier of lysine, amylose-lipid complex and starch-oil-lysine three-dimensional network can provide resistance to digestive enzymes. Annealing with corn oil and lysine can be a good prospect for the efficient modification of in vitro digestibility of starch.