Abstract: This study investigated the effects of adding bile acid and (or) cholesterol to low fishmeal diet on growth, serum biochemical and antioxidative parameters, hepatopancreas digestive enzyme activity and histology of Litopenaeus vannamei. First, a positive diet was formulated to contain 200 g/kg (FM20), then Clostridium autoethanogenum protein was used to substitute 100 g/kg fishmeal to form a low fishmeal diet containing 100 g/kg fishmeal (negative diet, FM10). Based on FM10 diet, cholesterol, bile acid, and their combination were added at the inclusion of 560 mg/kg, 150 mg/kg and 560 mg/kg + 150 mg/kg, respectively. The five isoproteic and isolipidic diets were fed to L. vannamei with initial body weight of (1.00±0.01) g for 56 days. Compared to the FM20 group, the weight gain, the activities of hemolymph lysozyme, hepatopancreas protease and lipase, and bile acid content in hepatopancreas and serum were decreased significantly (P<0.05), while feed conversion ratio, hemolymph alanine aminotransferase, aspartate aminotransferase activities and malondialdehyde content were increased in the FM10 group (P<0.05). In addition, the stellate structure of some hepatopancreas corpuscles disappeared, and the number of B and R cells decreased in the FM10 group. The individual addition of bile acids or cholesterol to FM10 diet tended to increase the WG, amylase activity and bile acid content in hepatopancreas and hemolymph (P>0.05). The combined supplementation of bile acids and cholesterol significantly improved the weight gain (+10.59%), lipase and protease activities, and decreased feed conversion ratio (−0.12) (P<0.05). Compared to the FM10 group, the hepatic tubules returned to normal and the stellate structure became clear when cholesterol and bile acid were added in diet. In conclusion, the addition of bile acid + cholesterol in low fishmeal diet improved the growth performance, digestive ability and protect the hepatopancreas health of L. vannamei. Bile acids and cholesterol showed a synergistic effect.