Microwave processing is a suitable technology for starch-based food processing. This work investigated the changes of structures and properties of high-amylose maize starch (HAMS) during short-time microwave irradiation (1–4 min). After 1 min of treatment, short amylopectin chains (DP 6–36) and intermediate amylose chains (DP 150–2000) of HAMS were partially broken down. Compared with native HAMS, treated HAMS (1 min) had the higher relative crystallinity, the intensity of the 9 nm lamellar peak, and fluorescence intensity under CLSM. Moreover, 1-min microwaving caused the lower viscosity and higher resistant starch content of HAMS. In the 2–4 min of treatment, the crystallinity, intensity of the lamellar peak and fluorescence intensity of HAMS granules decreased significantly, but no breakdown of starch molecule chains was observed, suggesting the realignment of the crystalline region during the process. Correspondingly, the viscosity increased and resistant starch content decreased. Our study provides a deeper understanding of the mechanistic effects of short-time microwave irradiation on high-amylose starch, which is of value for the processing of HAMS to produce novel functionality and nutritional values.