The objective of this study was to evaluate the nutritional value of Clostridium autoethanogenum protein (CAP) as a sole protein source at different dietary protein levels by growth performance, health status, and intestinal microbiota of grass carp. Grass carp (4.56 ± 0.01 g) were randomly divided into 6 treatments in triplicate (30 fish per replicate) and fed with gradient protein level (24.45%, 27.31%, 31.82%, 35.79%, 38.64%, and 42.82%, respectively; named CAP1, CAP2, CAP3, CAP4, CAP5, and CAP6) diets for 8 weeks. Final body weight (FBW) and specific growth rate (SGR) were the significantly highest in the CAP4 group (P < 0.05). Based on SGR and feed efficiency (FE), the dietary protein requirement for juvenile grass carp was estimated to be 36.14–37.90%. CAP4 promoted hepatopancreatic health by reducing lipid deposition and structural lesions, and improved the anti-inflammatory ability by upregulating the relative expression level of il10 (P < 0.05). At the same time, the CAP4 improved the length of mucosal fold and the thickness of muscular layer. In addition, CAP4 can maintain the composition and metabolic function of intestinal microbiota and enhance the endocrine system and bacterial chemotaxis. However, it is worth noting that increasing CAP inclusion depressed hepatopancreatic antioxidant capacity. In conclusion, CAP is a promising novel protein source for grass carp, while careful consideration of its side effect at high inclusion level is deserved.