The acute toxicity of nitrite on the mucosal morphology and oxidative stress response was investigated in the intestines of Litopenaeus vannamei after the exposure of 20 mg/L nitrite for 72 hr. The duration of nitrite stress influenced the shrimp survival, and the cumulative mortality rate was 36.7% at 72 hr. Hematoxylin and eosin (HE) stain showed that nitrite stress damaged the intestine connective and epithelium tissue. Specifically, nitrite stress increased the content of reactive oxygen species (ROS) production (O generation capacity), lipid peroxidation (LPO), and malondialdehyde (MDA). Meanwhile, the activities of antioxidant enzymes including superoxide dismutase (SOD) increased first, then catalase (CAT) and glutathione peroxidase (GPx) increased to the highest at 24 and 12 hr, respectively. The relative expression level of antioxidant genes including heat shock protein 70 (HSP70), ferritin, and thioredoxin (Trx) increased to the highest at 6, 12, and 48 hr respectively. After exposure to nitrite stress for 72 hr, the levels of antioxidant enzymes activity and genes expression all decreased. These results revealed that nitrite stress harmed the intestine barrier of L. vannamei by damaging the mucosal structure, inducing the oxidative stress, and confusing the antioxidant status.