Simple SummaryCholine is a vitamin-like nutrient and has many metabolic and physiological functions in aquatic animals. Unfortunately, the information on the optimal requirement of dietary choline in Litopenaeus vannamei is limited, and the molecular and metabolic mechanisms of choline on L. vannamei are unclear. Hence, in this study, the growth performance, whole-body composition, serum characteristics, hepatopancreatic antioxidant indexes, serum metabolome and hepatopancreas transcriptome were performed. In this study, the growth of L. vannamei was not affected by dietary choline. Dietary choline played an important role in arachidonic acid and glycerophospholipid metabolism and decreased the oxidant damage of L. vannamei, while excessive choline can inhibit the digestion of protein and reduce the whole-body crude protein in shrimp. Based on the results of weight gain and lipid peroxidation reduction, 1082 mg/kg dietary choline could meet the growth requirement of L. vannamei, but 2822 mg/kg dietary choline was needed to reduce peroxidation damage. The present study would provide valuable information on the requirement of choline in L. vannamei, and help to understand the molecular and metabolic mechanisms of choline in shrimp.To determine the response of Pacific white shrimp Litopenaeus vannamei to different levels of dietary choline, juvenile white shrimp (1.75 ± 0.09 g) were fed six semi-purified diets supplemented with 0 (control), 2000, 4000, 6000, 8000, and 12,000 mg/kg choline chloride for eight weeks. Growth performance, whole-body composition, serum characteristics and hepatopancreatic antioxidant indexes were evaluated. Meanwhile, serum metabolome and hepatopancreas transcriptome were performed to examine the overall difference in metabolite and gene expression. The weight gain, survival, specific growth rate, condition factor and hepatosomatic index were not affected by dietary choline levels. The shrimp fed 6000 mg/kg dietary choline chloride gained the maximal whole-body crude protein, which was significantly higher than that of shrimp fed with 12,000 mg/kg dietary choline. Serum total cholesterol of shrimp fed 6000 mg/kg dietary choline was higher than that in shrimp fed 4000 mg/kg choline. Dietary choline significantly decreased malondialdehyde content, superoxide dismutase, and glutathione peroxidase activities in shrimp hepatopancreas. Compared with the shrimp fed 6000 mg/kg dietary choline chloride, the glycerophospholipid metabolism pathway was significantly enriched in the shrimp fed 0 mg/kg dietary choline chloride, and the choline content and bile salt-activated lipase-like expression were upregulated. The expression of trypsin-1-like in protein digestion and absorption pathway was significantly downregulated in the shrimp fed 12,000 mg/kg dietary choline chloride. Apolipoprotein D might be a potential biomarker in shrimp, and dietary choline played an important role in lipid metabolism, especially in the reduction of oxidative damage in L. vannamei. Based on the results of weight gain and degree of oxidative damage, 1082 mg/kg dietary choline could meet the growth requirement of L. vannamei, but 2822 mg/kg dietary choline was needed to reduce peroxidation damage.