Traditional solvent-based approaches for the separation of protein from plant sources are widely used, although they can lead to low protein yield and protein degradation. In this study, protein isolation was conducted by alkaline extraction (AE) and isoelectric precipitation, and the effect of high-pressure homogenisation (HPH) (0 MPa [control], 50, 100, and 150 MPa) at two different stages during extraction was assessed, namely high pressure homogenisation followed by alkaline extraction (HPH + AE) or alkaline extraction followed by high pressure homogenisation (AE + HPH). Although the control procedure resulted in a protein yield of 45.99 ± 0.72%, the highest protein yield was determined by AE + 100 MPa HPH (61.92 ± 1.06%). Solubility, foaming capacity, emulsification index, hue angle, and denaturation enthalpy of the isolated protein increased after HPH treatments compared to control. Generally, the high-pressure treatment during protein isolation increased pumpkin seed press cake's protein yield and enhanced the isolates' techno-functional properties.