The present study was conducted to investigate the effects of reduced glutathione (GSH) supplementation in practical diet on growth performance, anti-oxidative response, disease resistance and intestine morphology of shrimp Litopenaeus vannamei. Two control diets based on the commercial formulation were designed with high level (27%) and low level (22%) of fish meal, respectively. Based on the control diet with low level of fish meal, 75, 150 and 225 mg/kg of GSH were added, respectively, to make the other three experimental diets. The five formulated diets were named as C1, C2, G1, G2 and G3, respectively. The shrimp (initial body weight: 0.30 ± 0.02 g) were fed with the five experimental diets for 8 weeks followed by a challenge test with Vibrio parahaemolyticus. The results showed that the specific growth rate (SGR) of shrimp in the C2 group was significantly lower than that in C1. The SGRs in G1 and G2 had no significant difference with those in C1 and C2. However, the SGR in G3 was significantly lower than that in C1. The serum GSH concentration in C2 was significantly lower than the other groups, but the malondialdehyde concentration was significantly higher. The supplementation of dietary GSH significantly improved the total anti-oxidative capacity and activities of glutathione S-transferase and glutathione peroxidase in serum. The villus height of intestine in the GSH supplemented groups had no significant difference with C1, but was significantly higher than C2. The jejunum wall thickness of intestine in G2 and G3 was significantly higher than those in the other groups. After the challenge test, the cumulative mortalities in G1 and G2 were significantly lower than C1 and C2. However, there was no significant difference in cumulative mortalities among G3, C1 and C2. In conclusion, based on the present experimental conditions, 75–150 mg/kg of GSH was suggested to be supplemented into the practical diet to improve the growth, anti-oxidative capacity, disease resistance and gut health of shrimp L. vannamei.