Rapeseed oil cake (ROC) is a biomass of particular interest for biorefinery and food activities, due to its high protein, lignin, polyphenol and fiber contents. An environmental separation process combining ultrafine milling and electrostatic separation was recently developed for proteo-lignocellulosic plant fractionation. Several of the parameters of the fractionation process (milling mode, electrode voltage, charge, particle size, etc.) can affect the efficiency of protein separation and extraction. We assessed the impact of three different mechanical modes of milling — ball milling, impact grinding and jet milling — on the electrostatic separation (ES) of lignin and protein from ROC biomass. The initial biomass and the ES fractions were characterized and correlations were identified between particle colors and sizes and their protein and fiber contents. Mechanical grinding had a significant impact on the surface properties of the particles, influencing their charge and the electrostatic separation of lignin and protein from biomass. The combination of impact grinding and electrostatic separation appear the most efficient and effective technologies for rapeseed oil cake protein concentrate