The effect of short-range molecular order of gelatinized starch on starch retrogradation was investigated by heating starch-water mixtures to different final temperatures in a differential scanning calorimeter (DSC) followed by storage at 4 °C for 7 days. The extent of residual short-range molecular order in the gelatinized starch, as determined by the area and intensity of the Raman bands at 480 and 1080 cm−1, decreased with increasing final DSC-heating temperature or increasing water content. The DSC enthalpy changes, long-range crystallinity and short-range molecular order of the retrograded starches increased initially to a maximum and then decreased with further decreases in short-range molecular order of the gelatinized starches. These results indicated that the extent of starch retrogradation increased initially and then decreased as the amount of residual short-range molecular order in the gelatinized starches decreased, and that retrogradation was most favored by an optimal amount of residual short-range molecular order in the gelatinized starch, which was determined by the heating temperature and water content. Retrogradation occurred to a lesser extent with lower or higher amounts of short-range molecular order in the gelatinized starches. This study provides new insights into starch retrogradation by showing that the state of glucan chains in gelatinized starch influences their retrogradation behavior.