Our previous study demonstrated that based on growth performance and feed utilization, cottonseed meal (CSM) could substitute 20% fishmeal (FM) without adverse effect on golden pompano (Trachinotus ovatus). Muscle deposition was also an important indicator to evaluate the efficiency of alternative protein sources. Therefore, the present study was conducted to explore the changes of physiobiochemical and nutrient metabolism in muscle after FM replaced by CSM. Four isonitrogenous and isolipidic experimental diets (42.5% crude protein, 14.0% crude lipid) were formulated to replace 0% (CSM0 diet), 20% (CSM20 diet), 40% (CSM40 diet), and 60% (CSM60 diet) of FM with CSM. Juvenile fish (24.8 ± 0.02 g) were fed each diet for 6 weeks. The results presented, which, compared with the CSM0 diet, CSM20 and CSM40 diets, had no effect on changing the muscle proximate composition and free essential amino acid (EAA) concentration. For glycolipid metabolism, the CSM20 diet did not change the mRNA expression of hexokinase (hk), glucose transport protein 4 (glut4), glucagon-like peptide 1 receptor (glp-1r), while over 20% replacement impaired glucose metabolism. However, CSM20 and CSM40 diets had no effect on altering lipid metabolism. Mechanistically, compared with the CSM0 diet, the CSM20 diet did not change muscle nutritive metabolism through keeping the activities of the nutrient sensing signaling pathways stable. Higher replacement would break this balance and lead to muscle nutritive metabolism disorders. Based on the results, CSM could substitute 20–40% FM without affecting the muscle nutritive deposition. All data supplemented the powerful support for our previous conclusion that CSM could successfully replace 20% FM based on growth performance.