This work aims to investigate the effect of different extraction and drying methods on pumpkin seed protein’s structure and functional properties. Pumpkin seed proteins (PSP) have been extracted from pumpkin seed meal (PSM), a by-product of pumpkin seed oil processing. The extraction of PSP from PSM by alkaline extraction (AE) and enzymatic extraction (EE), followed by freeze drying (FD) and spray drying (SD), revealed differences in the physicochemical and functional properties of PSP. PSP obtained by EE-SD exhibited higher α-helix contents while the EE-FD sample had lower α-helix contents. Compared to AE-PSP, EE-PSP exhibited higher solubility. EE-SD had higher solubility, while AE-FD had higher FP. Understanding the effects of extraction and drying methods on structural and functional properties could help to select an appropriate method to optimize the utilization of PSP in the food industry.