The study estimated the effect of substituting fishmeal with cottonseed protein concentrate (CPC) in practical diets on growth performance, flesh quality and metabolomics of largemouth bass (Micropterus salmoides). In a basal diet with fishmeal inclusion of 400 g/kg, five diets were formulated with CPC inclusion to isoproteicly replace 0%, 15%, 30%, 45% and 60% of dietary fishmeal, correspondingly, the fishmeal inclusion was 400 g/kg (FM40), 340 g/kg (FM34), 280 g/kg (FM28), 220 g/kg (FM22), 160 g/kg (FM16), respectively. Then, the five diets were fed to largemouth bass with initial body weight of 75.0 g for 12 weeks. The results showed that 15% and 30% of dietary fishmeal could be successfully replaced by CPC without significant effects on the weight gain (WG) and feed conversion ratio (FCR), while the WG was decreased and FCR was increased when 45% and 60% of dietary fishmeal were replaced by CPC (P < 0.05). With the increase of replaced fishmeal level by CPC, the flesh hardness and the content of heat-insoluble collagen showed a significantly negative linear trend (P < 0.05), while flesh drip loss (at 12th h and 24th h), n-6 polyunsaturated fatty acid, total free amino acids and flavor amino acids contents showed a significantly positive linear trend (P < 0.05). FM22 and FM16 groups showed lower flesh hardness and higher drip loss (24th h) than FM40 group (P < 0.05). In muscle histology and metabolomics, FM16 group presented higher myofiber density and lower myofiber diameter than FM40 group. A total of 97 differential metabolites were detected between FM16 and FM40 groups, which mainly enriched in pathways as biosynthesis of amino acid, protein digestion and absorption, D-arginine and D-ornithine metabolism, histidine metabolism, alanine, aspartate and glutamate metabolism. Conclusively, in a practical diet containing 400 g/kg fishmeal, CPC could replace 30% fishmeal (120 g/kg) without significant impacts on growth, flesh composition, texture, flavor and muscular tissue structure, and high fishmeal replacement (≥ 45%) decreased growth performance and flesh quality of largemouth bass.