A 10-week feeding experiment was performed to determine the impacts of partial substitution of soybean meal (SB) with pumpkin seed cake (PSC) in Oreochromis niloticus diets on water quality, growth rate, antioxidant capacity, immunity, and carcass composition. One hundred and fifty tilapia fish (average weight, 11.93 ± 0.17 g) were randomly allocated to five diets. The first diet (the basal diet) contained 420 g of SB per kg of feed. The remaining four diets, namely, D1, D2, D3, and D4, had SB partially replaced by PSC at 10%, 20%, 30%, and 40%, respectively. The results revealed that D4 and D1 significantly improved dissolved oxygen levels, while water temperature, pH, total ammonia, and nitrate levels were not significantly affected. Replacing SB with PSC significantly improved specific growth performance indicators and feed conversion compared to the control, with the D4 group showing the best values. Increasing PSC levels decreased serum glucose, aspartate aminotransferase, alanine aminotransferase, cholesterol, and triglyceride levels. In contrast, the D4 group had higher globulin, albumin, total protein, and lysozyme serum levels. Moreover, fish-fed PSC had significantly increased superoxide dismutase, glutathione peroxidase, and catalase activities and significantly decreased malondialdehyde levels. Increasing PSC substitution levels in fish diets increased the ash and crude lipid contents in the bodies of the fish, while crude protein and moisture decreased. In conclusion, replacing SB with PSC in fish diets significantly enhances growth performance, feed conversion, and fish health. Moreover, the findings suggest that PSC can be a promising alternative protein source for sustainable aquaculture practices.