This study investigated the effects of annealing on the structural and physicochemical properties of rice starch below the onset temperature (To) by 5 °C and 15 °C. The results revealed that annealing improved the gelatinization temperature of rice starch, decreased the swelling power, solubility, and paste viscosity of rice starch, and had no significant effects on the morphological structure and crystal configuration of rice starch. In one-step annealing, the annealing temperature of 60 °C is more conducive to the rearrangement of starch molecules, so its crystallinity, short-range ordered structure, and gelatinization temperature are higher than at 50 °C; however, its RDS, SDS, and RS contents will be increased. During the two-step annealing treatment, the temperature change is not conducive to the molecular chain rearrangement and to the formation of perfect crystalline structure, which increases the sensitivity of enzymes to starch, so the RDS content of starch increases significantly, while the RS content decreases.