AbstractBackground and ObjectivesPulsed electric field (PEF) treatment is currently a hot spot in food science research. The improvement and regulation of protein structure and physicochemical properties after PEF treatment are a new development in its processing technology. This study aimed to clarify the effects of moderate‐intensity PEF treatment on the structure and physicochemical properties of foxtail millet (Setaria italica) prolamin and its action rules.FindingsPEF treatment changed the spatial conformation of millet prolamin, increased its hydrophobicity and solubility in alcohol solution, but did not change the subunit composition distribution. It also promoted the increase in soluble aggregates, resulting in an increase in protein particle size. The α‐helix content of the protein decreased, the β‐sheet content increased, the number of disulfide bonds increased, and the protein structure became more orderly after treatment.ConclusionsThe results showed that the structure and physicochemical properties of millet prolamin significantly changed after the moderate‐intensity PEF treatment.Significance and NoveltyThe increased hydrophobicity of prolamin makes it a strong future candidate in medicinal excipients, food packaging materials, and biodegradable polymers.