The understanding of the protein structure-function relationship is very important to the study of protein chemistry. In this research, the physicochemical, functional, and structural properties of the storage protein fractions from two defatted proso millet cultivars (Dawn and Plateau) were determined and reported. The results show that protein recovery efficiency of 53.5% and 60.1% was recorded for Dawn and Plateau, respectively. The average denaturation temperature of all fractions was about 82.1 ± 3.5 °C. Surface hydrophobicity values for Dawn fractions were 11781, 10594, 316, and 2225 for albumin, globulin, and glutelin, respectively and 3415, 2865, 353, and 456 for Plateau fractions, respectively. Most of the protein fractions showed the highest solubility at pH 9 and lowest solubilities at pH ≤ 7 with solubility range from 5.7 to 100%. Emulsifying activity index (EAI) of less than 0.25 m2/g was recorded for most fractions, while the highest emulsion stability index (ESI) recorded was about 60 min. Prolamin fractions showed three major peptide bands of 11, 14, and 24 kDa while glutelin fraction revealed only a major band of 15 kDa and several minor bands of 11, 22, 24, 78, 209 kDa. No differences in the electrophoresis pattern were observed for the fraction with or without a reducing agent. Extraction and characterization of proso millet protein fractions. Display omitted •Prolamin is the major protein fraction in proso millet and with least solubility.•Albumin and globulin showed a higher S0 to prolamin and glutelin protein fractions.•EAI was highest in albumin fraction while ESI was highest in prolamin fraction.•FC and FS is highest in glutelin fraction but lowest in prolamin fraction.•Major polypeptide bands in prolamin are 11, 14 and 24 kDa; 15 kDa for glutelin.