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Simple Summary: With the development of the breeding industry, the production of fishmeal will not be able to meet the needs of feed production in the future. Clostridium autoethanogenum protein meal (CAP) is a by-product of people using carbon monoxide exhaust from the steel industry to produce ethanol, with a potential to become a substitute ingredient for fishmeal to alleviate the shortage of fishmeal. This study was conducted on pearl gentian grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂), an economic fish species widely cultured in Southeast Asia. After using different concentrations of CAP to replace fishmeal in the feed for eight weeks, respectively, we found that it could replace 45% of fishmeal in the pearl gentian grouper feed through growth performance, various physiological and biochemical indexes analysis experiments, but a 60% replacement level would significantly affect health and growth. These findings provide a reference for the promotion and further research of CAP as a new environmentally friendly ingredient, which is positive for the sustainable development of the farming industry and even for the utilization of waste gas from the steel industry. In this study, we present data from an eight-week growth trial with pearl gentian grouper fed either a reference diet (FM) with a fishmeal level of 50%, or test diet wherein 15% (CAP15), 30% (CAP30), 45% (CAP45), and 60% (CAP60) fishmeal was replaced by Clostridium autoethanogenum protein meal (CAP). Results showed that the weight gain and daily feed intake ratio of CAP60 were significantly lower than the FM group. In the serum, compared to the FM group, the content of malondialdehyde (MDA), the activities of alanine aminotransferase in CAP60 and CAP45 groups, and acid phosphatase in the CAP60 group were significantly higher, while the content of total cholesterol in CAP60 and CAP45 groups was significantly lower. In the liver, compared to the control group, the content of MDA in the CAP60 group was significantly higher. 3-hydroxy-3-methylglutaryl coenzyme A reductase in CAP30 to CAP60 groups and farnesoid X receptor in CAP60 were significantly upregulated. In distal intestines, the activities of trypsin and superoxide dismutase of CAP30 to CAP60 groups were significantly lower than the FM group. In conclusion, for pearl gentian grouper, CAP could replace up to 45% of the fishmeal in the feed, while a 60% replacement level will affect cholesterol bile acid metabolism and health. [ABSTRACT FROM AUTHOR]