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The purpose of this study was to develop the potential of cottonseed protein concentrate (CPC) and Clostridium autoethanogenum protein (CAP) in the diet of rainbow trout (Oncorhynchus mykiss) by evaluating the effects of substituting fishmeal with a CPC and CAP mixture on growth performance, nutrient utilization, serum biochemical indices, intestinal and hepatopancreas histology. In a basal diet containing 200 g/kg fishmeal (Con), the mixture of CPC and CAP (1:1) was used to reduce dietary fishmeal to 150, 100, 50 and 0 g/kg, to form five diets with the same crude protein and crude lipid contents (CON, FM-15, FM-10, FM-5 and FM-0). Then, the five diets were fed to rainbow trout (35.00 ± 0.05 g) for 8 weeks. The weight gain (WG) and feed conversion ratio (FCR) of the five groups were 258.72%, 258.82%, 249.90%, 242.89%, 236.57%, and 1.19, 1.20, 1.24, 1.28, 1.31, respectively. FM-5 and FM-0 groups showed significantly lower WG and higher FCR than the CON group (p < 0.05). In terms of whole-body composition, such as moisture, crude ash, and crude protein, no significant difference was observed among all the groups (p > 0.05), except that significantly higher crude lipid content was detected in the FM-0 group than in the CON group (p < 0.05). In the FM-5 and FM-0 groups, protein efficiency, protein retention, intestinal protease activity and amylase activity were significantly lower than in the CON group (p < 0.05). Compared to the CON group, the serum contents of glucose and total cholesterol in the FM-0 group as well as MDA in the FM-5 and FM-0 groups were significantly increased, and catalase, superoxide dismutase, and total antioxidant capacity were decreased (p < 0.05). In intestine and hepatopancreas histology, the intestinal villus height in the FM-5 and FM-0 groups and villus width in the FM-0 group were decreased significantly (p < 0.05), while no significant difference in hepatopancreas morphology was observed among all the groups except that some vacuolization was observed in the FM-0 group (p > 0.05). In summary, the mixture of CPC and CAP can effectively replace 100 g/kg fishmeal in a diet containing 200 g/kg fishmeal without adverse effects on the growth performance, nutrient utilization, serum biochemical, or intestinal and hepatopancreas histology of rainbow trout.