The structural and physicochemical characteristics of oil bodies from hemp seeds were explored in this study. Oil bodies from several plant-based sources have been previously studied; however, this is the first time a characterisation of oil bodies from the seeds of industrial hemp is provided. The morphology of oil bodies in hemp seeds and after extraction was investigated using cryo-scanning electron microscopy (cryo-SEM), and the interfacial characteristics of isolated oil bodies were studied by confocal laser scanning microscopy (CLSM). Proteins associated with oil bodies were characterised using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The effect of pH and ionic strength on colloidal properties of the oil bodies was investigated. Oil bodies in hemp seeds appeared spherical and sporadically distributed in the cell, with diameters of 3 to 5 μm. CLSM images of isolated oil bodies revealed the uniform distribution of phospholipids and proteins at their interface. Polyunsaturated fatty acids were predominant in the lipid fraction and linoleic acid accounted for ≈61% of the total fatty acids. The SDS-PAGE analysis of washed and purified oil bodies revealed major bands at 15 kDa and 50-25 kDa, which could be linked to membrane-specific proteins of oil bodies or extraneous proteins. The colloidal stability of oil bodies in different pH environments indicated that the isoelectric point was between pH 4 and 4.5, where oil bodies experienced maximum aggregation. Changes in the ionic strength decreased the interfacial charge density of oil bodies (ζ-potential), but it did not affect their mean particle size. This suggested that the steric hindrance provided by membrane-specific proteins at the interface of the oil bodies could have prevented them from flocculation at low interfacial charge density. The results of this study provide new tertiary knowledge on the structure, composition, and colloidal properties of oil bodies extracted from hemp seeds, which could be used as natural emulsions or lipid-based delivery systems for food products.