The objective of the present study is to enhance the resistant starch (RS) content of high amylose rice starch with heat–moisture treatment (HMT) for industrial application. The optimized HMT condition for achieving the highest RS content established using response surface methodology (RSM) was a temperature of 100 °C, moisture content of 24.2%, and a time of 11.5 h. Upon HMT, the RS content increased from 32.1% for native starch to 46.4% in HMT starch with optimized condition. HMT of the starches reduced the solubility and swelling power. The surface of HMT starch granules was more irregular than native starch. The X-ray diffraction (XRD) peak intensity at 2θ = 5° was greatly reduced by HMT, and the peaks at 22.7° and 24.2° were merged. HMT increased the gelatinization temperature and reduced the gelatinization enthalpy. HMT provides a method for the production of high-yield RS2 with high amylose rice starch in industrial application.