A feed trial was conducted in Litopenaeus vannamei to evaluate the effects of glycerol monolaurate (GML) supplement on growth, muscle amino acids, non-specific immunity, disease resistance, and intestinal flora structure. Juvenile L. vannamei [body weight = (0.31±0.02) g] were fed for 56 days with six iso-nitrogenous and isoenergetic experimental diets containing 0 (control group), 500, 1 000, 1 500, 2 000 and 2 500 mg/kg GML, respectively. The results showed that, ① 2 000 mg/kg GML significantly increased the weight gain rate and specific growth rate and significantly reduced the feed coefficient compared to the control group. ② Feeding L. vannamei with GML diet had no significant effect on the body composition of the fish. ③ L. vannamei fed with GML diet had significantly higher content of flavor amino acids and total amino acids. ④ Serum total protein content was significantly higher, LDL cholesterol content and glutathione transaminase activity were significantly lower in all addition groups; triglyceride content was significantly lower in those fed with 500 mg/kg and 1 500 mg/kg GML. ⑤ Superoxide dismutase activity and catalase expression were significantly up-regulated in those fed with 1 500 mg/kg GML, and IMD, Toll expressions, and lysozyme activity were significantly up-regulated in those fed with 2 000 and 2 500 mg/kg GML, and phenol oxidase pro expression was significantly up-regulated in those fed with 2 000 mg/kg GML. ⑥ All addition groups had significantly increased Chao1 and Ace indexes, L. vannamei fed with 1 500 and 2 500 mg/kg GML had significantly decreased Shannon index, and Simpson index was significantly decreased in those fed with 2 500 mg/kg. In summary, the results of the present study emphasized the favorable effects of adequate dietary GML on growth performance and non-specific immunity and intestinal flora. Dietary GML at 2 142.99 mg/kg GML was recommended to maintain optimal growth performance ofL. vannamei.