Clostridium autoethanogenum protein (CAP) is a safe and effective alternative protein source originated from the inactivated bacterial biomass parallelly produced with ethanol. However, there are very limited studies on the application of CAP in aquafeeds. The present study investigated the physicochemical and functional properties of CAP; the effects of dietary CAP levels and processing parameters on the physical properties of low-starch extruded floating feed; the potential mechanisms for the dietary CAP inclusion to improve the physical properties of extruded feed; the effects of dietary CAP levels on the growth performance and health conditions of largemouth bass (Micropterus salmoides). Firstly, we compared the differences in physicochemical and functional properties of CAP and low-temperature steam dried fish meal (LTFM). Extrusion trials were performed to investigate the effects of graded CAP substitution levels (replacing 0%, 25%, 50%, 75% and 100% of FM in the diet, named as CAP0, CAP25, CAP50, CAP75 and CAP100, respectively), preconditioning moisture content (28% and 32%) and screw speed (200 rpm and 300 rpm) on the physical qualities of low-starch extruded floating feed. The results showed that CAP had high crude proteins (832 g/kg), low crude lipids (19.0 g/kg) and low ash (35.0 g/kg) contents. The water-holding capacity, foaming capacity and foaming stability of CAP were significantly higher than those of LTFM (P < 0.05). Both LTFM and CAP solutions exhibited a pseudo-plastic behaviour. Both storage modulus (G’) and the loss modulus (G’’) of the CAP solution increased with the increase in temperature. The floatability of all extruded feeds reached 100%. As the replacement level of CAP increased, the peak viscosity of the unprocessed stuffs increased significantly (P < 0.05), whereas the pasting temperature decreased significantly (P < 0.05). Specific mechanical energy significantly increased with the increase in CAP substitution level (P < 0.05) and decreased with the increase in screw speed and moisture content (P < 0.05). Dietary CAP inclusion could improve the expansion degree, texture, oil absorption capacity and water stability of the extruded feeds. Moisture content and screw speed also had significant effects on the physical qualities of the extruded feed, and the recommended process parameters for the test diets were 28% moisture content and 300 rpm screw speed. Then, the extruded feeds produced under the processing parameters recommended in the previous chapter were used for further analysis. The thermal transition, protein-protein interactions, molecular weight distribution and secondary structure of the proteins of the unprocessed stuffs and extruded feeds were investigated. The results showed that the enthalpy ( △ H) of starch in the unprocessed CAP75 and CAP100 groups were significantly higher than that in the CAP0 group (P < 0.05), whereas the △ H of starch in the extruded CAP25 and CAP75 groups were significantly lower than that in the CAP0 group (P < 0.05). The amount of proteins bonded by disulfide bonds and interactions between hydrogen bonds and disulfide bonds significantly decreased in CAP inclusion feeds (P < 0.05). The absolute content of the water-soluble protein in unprocessed stuffs decreased as the amount of CAP replacement increased. After extrusion, the relative content of higher-molecular-weight fraction (110-1000  103 g/mol) in the CAP50 (9.28%) and CAP75 (12.67%) groups was higher than that of the control group (6.12%), while the higher-molecular-weight fraction disappeared in the CAP100 group. In unprocessed stuffs, the β-sheet ratio increased with the increase in the amount of CAP replacement and the β-sheet ratio in the CAP100 group was significantly higher than that of the control group (P < 0.05). The α-helix to β-sheet ratio decreased in the CAP inclusion groups. In extruded feeds, the α-helix ratio and β-turn ratio in the CAP inclusion groups were significantly higher than that in the control group (P < 0.05), while the β-sheet ratio decreased significantly at the same time. The α-helix to β-sheet ratio increased significantly from 0.61 to 0.72 (P < 0.05). Finally, an in vitro digestion experiment and an 8-wk growth experiments were conducted to evaluate the molecular weight distribution of the CAP hydrolysate, and the effects of dietary CAP levels on the growth performance, plasma parameters, hepatic and intestinal health, and the diversity of gut-adherent microbiota of largemouth bass. The fish (initial body weight of 47.99 ± 0.01 g) were fed diets where CAP gradually replaced 0 (CAP0), 12.5 (CAP12.5), 25 (CAP25), 37.5 (CAP37.5) and 50% (CAP50) of LTFM in the diet. Results showed that the content of peptides below 1000 Da in the CAP hydrolysate (0.56 mg/mL) was higher than that of the LTFM hydrolysate (0.48 mg/mL). Dietary CAP inclusion had no negative effect on growth performance, while whole-body lipid content significantly reduced in the CAP25 and CAP50 groups (P < 0.05). The plasma alanine aminotransferase (ALT) activities and triglyceride (TG) concentrations in the CAP inclusion groups were significantly lower than those in the CAP0 group (P < 0.05). The richness and diversity of the gut-adhesive microbiota in the CAP50 group were significantly higher than those in the CAP0 group (P < 0.05). Dietary CAP inclusion inhibited inflammatory responses by down-regulating the mRNA levels of interleukin 1β (IL1β), interleukin 10 (IL10) and transforming growth factor β1 (TGFβ1) (P < 0.05) in the liver. The mRNA levels of acetyl-CoA carboxylase 1 (ACC1) were significantly down-regulated in the CAP12.5, CAP25 and CAP37.5 groups (P < 0.05), while that of fatty acid synthase (FASN) was significantly down-regulated in the CAP50 group (P < 0.05). The present research showed that dietary CAP inclusion could improve the physical properties of low-starch extruded floating feed by promoting proteins refolding and aggregation between protein and starch during the extrusion process. Besides, dietary CAP inclusion could improve the hepatic and intestinal health of largemouth bass. Overall, CAP is a promising protein substitute that can be used in aquaculture.