The present study aimed to examine the effects of short-term exposure to ammonia on stress and oxidative responses in shrimp (Litopenaeus vannamei) and to determine whether the antioxidant system related to the regulatory role of transcription factors and stress proteins was activated. Shrimp were exposed ammonia-N at four concentrations: 0 (control), 5, 10, and 15 mg/L, for 48 h. The hepatopancreas was sampled to measure the levels of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO); the activities of superoxide dismutase (SOD), catalase (CAT), nitric oxide synthase (NOS); and the expression levels of GSH-px (encoding glutathione peroxidase), GST (encoding glutathione-S-transferase), HSP70 (encoding heat shock protein 70), HSP90 (encoding heat shock protein 90), p53, RELISH, and AKIRIN. We observed that exposure to a high ammonia content increased the abundance of oxidative factors (MDA, CAT, SOD, NOS, and NO), reduced the levels of GSH, and upregulated the mRNA expression levels of antioxidant genes (GSH-px and GST), stress-related genes (HSP70 and HSP90), and transcription factor genes (p53, RELISH, and AKIRIN). These results indicated that ammonia induced oxidative stress and inflammation. Both enzymatic and nonenzymatic antioxidant defense systems are involved, which might be regulated by HSPs, as well as certain transcription factors, such as p53 and nuclear factor kappa B (NF-κB), thus mounting an adaptive response to help rebalance redox homoeostasis.