Abstract: In this paper, hemp protein isolate (HPI) of four different varieties was extracted by alkali dissolution and acid precipitation. The molecular weight of HPI was determined and its structures were characterized by circular dichroism, free sulfhydryl and disulfide bond content and surface hydrophobicity. Finally, its functional properties were determined and the correlation analysis between functional properties and spatial structure was conducted. The results showed that HPI was mainly composed of edestin (~50 kDa) and albumin (10~15 kDa), and the former had two subunits. Random coil accounted for about 50% of the secondary structure. The content of disulfide bond in HPI from Dongbei was (5.20±0.32) μmol/g which means a more stable structure, and the content of free sulfhydryl groups HPI from Shanxi was (15.18±0.32) μmol/g that means it was easier to form aggregates. At pH4~6, the solubility of HPI was only 4.85%~31.48%. The water holding capacity and oil holding capacity of HPI were 3.26~4.24 and 4.36~6.03 g/g, respectively, superior to most plant proteins. Compared with other plant proteins, HPI had poor emulsifying and foaming capacity. Alpha-helix and surface hydrophobicity were significantly positive in correlation with emulsifying ability (r=0.70, 0.65, P<0.05). There was highly significant positive correlation between emulsifying stability and random coil (r=0.74, P<0.01), and distinct negative correlation between emulsifying stability and disulfide bond (r=−0.72, P<0.01). Overall, the functional properties and spatial structure of different varieties of HPI were quite different. The research results could provide a theoretical basis for the intensive processing of HPI and its application in different foods and formulas.