This study investigated the effects of protein-restricted feeding regimes on the growth, body composition, hemolymph parameters, digestive and serological enzyme activities, immune and antioxidant system responses, and relative gene expression in juvenile whiteleg shrimp (Penaeus vannamei) with an initial weight of 0.28 ± 0.03 g. The experimental groups were Control (feeding with dietary 40% protein (40P)), 2P401P35 (2 days feeding with 40P and 1 day feeding with 35P), P40P35 (1 day feeding with 40P and 1 day feeding with 35P), 1P402P35 (1 day feeding with 40P and 2 days feeding with 35P), and 35%P (feeding with 35P). The results showed no difference in weight gain and feed efficiency among the Control (10.27 g, 64.3%) and 1P402P35 (9.63 g, 61.26%) groups. Also, survival rate, protein efficiency ratio, lipid efficiency ratio, and productive protein value did not differ between these two treatments. The only negative side of the 1P402P35 group was lower protein content in the body than in the Control. Also, the 1P402P35 treatment had insignificant values of hemolymph parameters, immunity, antioxidant system parameters, serological enzymes, and gene expression compared to the Control. Also, this study revealed that 35% protein is not by far enough to provide maximum growth and health for whiteleg shrimp. In conclusion, applying daily protein restriction of 1 day feeding with 40P and 2 days feeding with 35P is recommended to reduce protein usage and improve water quality in this species. The effects of protein restriction (one day feeding with dietary 40% protein and followed by two days feeding with dietary 35% protein and using this cycle for 56 days) and FM replacement with an alternative protein mixture (yellow mealworm, spirulina, rice concentrate; 4:1:4.5 ratio) on growth, body composition, hemolymph parameters, digestive enzymes, serological enzymes, immune and antioxidant system, and relative genes expressions in whiteleg shrimp (Penaeus vannamei) (0.63±0.04 g)