As demand for sustainable plant protein rises, pumpkin seeds emerge as a promising but underutilized source. Conventional alkaline extraction (ALK) often impairs protein functionality, prompting interest in non-thermal alternatives. This study systematically compared the functional, colloidal, and structural properties of pumpkin seed protein isolates obtained via ALK (conducted at 50 °C), ultrasound-assisted (UAE), and microwave-assisted extraction (MAE). UAE produced the highest extraction yield (50.07%), superior overall solubility, greatest water and fat absorption capacities, and lowest least gelation concentration (12%). Furthermore, UAE best preserved native protein secondary structure (retaining 43.45% alpha-helix), as quantified by FTIR peak deconvolution, and maintained an intact, flake-like morphology under scanning electron microscopy (SEM), yielding the most uniform particle size distribution. Conversely, MAE achieved the highest protein content (73.53%) and the most negative zeta potential, leading to the highest emulsifying and foaming capacities despite inducing a bimodal particle size and irregular, porous surface morphology. ALK performed the poorest across structural and functional metrics, severely denaturing the proteins due to combined alkaline and thermal stress. UAE is recommended for applications requiring optimal solubility and gelation, whereas MAE is highly effective for emulsion- and foam-based food systems, reinforcing pumpkin seeds as a viable sustainable protein ingredient.