To increase the value of pumpkin seed meal and investigate the activity and mechanism of pumpkin seed protein peptides in delaying muscle atrophy, pumpkin seed protein-derived peptides were prepared from pumpkin seed meal using enzymatic hydrolysis. The preparation process was optimized, and the ability of pumpkin seed protein peptides to delay muscle atrophy was evaluated. Different proteases were employed to hydrolyze pumpkin seed meal, and proteinase screening, single-factor experiments, and response surface methodology (RSM) experiments were carried out. By evaluating the effects of the enzymatic hydrolysis products on oxidative stress, inflammation, and mitochondrial function, the mechanism of muscle atrophy retardation was explored. Pumpkin seed protein peptides prepared via dual-enzyme complex hydrolysis using alkaline protease and trypsin displayed the most potent effects. The optimal process conditions included 3.50 hours of hydrolysis, an alkaline protease to trypsin ratio of 2.97:1, and an enzyme dosage of 0.76%. Pumpkin seed protein peptides prepared under these conditions could increase the Caenorhabditis elegans body bending frequency to 20.63 times/30 s, significantly greater than that of the non-intervention group (13.30 times/30 s). Pumpkin seed protein peptides under optimal conditions also exhibited excellent antioxidant activity (a 2.50-fold increase in CAT activity, a 0.49-fold increase in GSH activity, and a 75.21% reduction in MDA level), anti-inflammatory activity (a 41.95% reduction in TNF-α and a 69.57% reduction in IL-6), and mitochondrial function (a 1.10-fold increase in ATP). This study identified a satisfactory process to prepare pumpkin seed protein peptides and confirmed the ability of pumpkin seed protein peptides to improve locomotion in nematodes and enhance the antioxidant, anti-inflammatory, and mitochondrial function of C2C12 cells. These findings provide a theoretical basis for the use of pumpkin seed protein peptides to delay muscle atrophy.