In this study, the effects of preheated treatments on physicochemical properties of resistant starch type III formation by pullulanase hydrolysis of High Amylose Rice Starch (HARS) were investigated. A debranching enzyme (Pullulanase, 8 U g-1 starch at 55°C for 0-48 h) was introduced to modify the amylopectin molecules of 15% (w/w) HARS suspension (32.10%, amylose content) which had been preheated at 95 and 121°C for 30 min. Retrogradation gels of debranched starches with different degrees of hydrolysis (0.14 to 3.10%) were then induced at 4°C for 16 h. Afterward, one cycle of the freeze-thaw process (-10/30°C) was applied to promote syneresis of the retrograded starches. Results show that pullulanase hydrolysis enhanced the degree of syneresis (33.22, 45.27 and 58.91% for non-debranched and debranched starches which had been preheated at 95 and 121°C for 48 h, respectively). The debranched starches with higher degree of hydrolysis provided products with higher resistant starch contents. The resistant starch content increased quadrupled with debranching and the freeze-thaw process (4.07 to 10.68% and 5.12 to 19.32% for 0 to 48 h pullulanase hydrolysis of HARS preheated at 95 and 121°C, respectively). Results had shown that after debranching and retrogradation, the HARS molecules had rearranged and changed their crystal pattern from A to V-type pattern, as revealed by X-ray diffraction analysis. In vitro starch hydrolysis index of the RS III samples from 0 to 48 h of pullulanase hydrolysis of the HARS which had been preheated at 95 and 121°C were reduced from 71.591 to 41.69% and 68.66 to 26.83%, respectively.