To investigate the effect of electron beam irradiation (EBI) pretreatment on the multiscale structure and physicochemical properties of esterified starch, this study used EBI pretreatment to prepare glutaric anhydride (GA) esterified proso millet starch. GA starch did not show the corresponding distinct thermodynamics peaks. However, it had a high pasting viscosity and transparency (57.46–74.25 %). EBI pretreatment increased the degree of glutaric acid esterification (0.0284–0.0560) and changed its structure and physicochemical properties. EBI pretreatment disrupted its short-range ordering structure, reducing the crystallinity, molecular weight and pasting viscosity of glutaric acid esterified starch. Moreover, it produced more short chains and increased the transparency (84.28–93.11 %) of glutaric acid esterified starch. This study could offer a rationale for using EBI pretreatment technology to maximize the functional properties of GA modified starch and enlarge its implementation in modified starch. •It's a comprehensive system for the multiscale - functionality of proso millet starch.•GA esterification was explored to fill the gap of polyacid esterification.•EBI pretreatment improved the degree of substitution of esterification.•EBI and GA enhanced the transparency of proso millet starch.