Recently, there are considerable interests in the influence of prolamins on eating quality of grains. To inquire the potential effect of prolamins on the palatability of foxtail millet, prolamin characteristics under its raw (PR) and post-cooked (PC) state among three typical varieties with high (Zhonggu, ZG), medium (Zhaonong, ZN), and low (Hongmiao, HM) palatability were compared. The distinctive differences in amino acid composition, molecular structure, physicochemical properties were found in PRs and PCs, especially for HM variety. HM-PR recorded the lowest hydrophobic amino acids and surface hydrophobicity while having the superior hydration properties. The lowest denaturation temperature was found in HM-PR, which also had the highest denaturation enthalpy (ΔH). Nevertheless, HM-PR exhibited irregularly spherical protein body with the largest mean diameter. Evidenced by the highest random coil and lower α-helix and β-sheet content, a less stable secondary structure of HM-PR was found, corresponding to the most intensified disulfide cross-linking and protein aggregations in HM upon cooking. Overall, HM-PR was presumed to greatly affect the hydro-thermal utilization efficiency of starch granules during cooking, given the steric-hindrance effect of prolamins on granules in endosperm. The Present study provided new insights into the role of prolamins on foxtail millet palatability.