To evaluate the feasibility of cottonseed protein in largemouth bass (Micropterus salmoides) diet, five isonitrogen-isolipid experimental diets (CPC₀ as control group, CPC₁, CPC₂, CPC₃, and CPC₄) were prepared by replacing 30% fish meal with 4 cottonseed proteins of different quality. Micropterus salmoides [average body weight (12.20±0.11) g] were fed in indoor circulating culture system for 8 weeks. The results showed that the nutrient composition, contents of gossypol, raffinose and stysaccharide of the four kinds of cottonseed proteins were different, and CPC₃ cottonseed protein quality was the best. The final body weight, weight gain rate and specific growth rate of CPC₃ group were significantly higher than those of the other groups, but there were no significant difference in the routine nutrients and muscle amino acid nutritional composition among all groups. The activities of SOD and GSH-Px and the mRNA expressions of CAT and SOD in liver of CPC₃ group were the highest, while the content of MDA was the lowest. The relative expressions of anti-inflammatory factors IL-10 and TGF-β were the highest in CPC3 group, while the relative expressions of pro-inflammatory factors IL-1β, IL-8 and TNF-α were the lowest in CPC₃ group. In addition, cottonseed protein sources could significantly affect the liver protein metabolism of largemouth bass, and the activities of ALT and AST and the expression levels of P13K, Akt, m-TOR, S6K1 and 4E-BP in CPC₃ group were the highest. Meanwhile, it was found that CPC₃ group had the highest intestinal T-SOD enzyme activity, the lowest MDA content, and the lowest intestinal permeability indexes (diamine oxidase, D-lactic acid and endotoxin). Cottonseed protein sources also affected the expression of tight junction protein-related genes ZO-1, Claudin-1 and Occludin in the intestine. The results showed that different cottonseed protein quality had different effects on the growth and health of M. salmoides, among which cottonseed protein CPC₃ showed the best effect and significantly improved the liver and intestinal health of M. salmoides, thereby promoting the growth of M. salmoides. Cottonseed protein CPC₃ can be used as a good protein source for M. salmoides feed.