This study evaluated the acute toxicity and sublethal effects of nitrite on oxidative stress parameters in early juvenile Brazilian flounder, Paralichthys orbignyanus. Fish were exposed to six concentrations of nitrite: 20, 40, 80, 170, 330 and 550 mg NO2−N-L−1, plus a control with no added nitrite, all in triplicate for 96 h. Every 24 h, the tanks were monitored to check possible mortality to estimate the LC50. At the end of the experiment, fish were sampled for stress oxidative analysis. All fish exposed to 550 mg NO2−N-L−1 died within the first 24 h. The LC50 ranged from 427.8 to 393.1 mg NO2−N-L−1 for 24 and 96 h respectively. The total antioxidant capacity against peroxyl radicals (ACAP) showed a gradual reduction in fish exposed to nitrite levels from 170 mg NO2−N-L−1. The glutathione S-transferase (GST) activity was higher in 80, 170 and 330 mg NO2−N-L−1. The non-proteic (NPSH) and proteic (PSH) thiols levels were higher in 170 and 330 mg NO2−N-L−1 compared with the control, 20 and 40 mg NO2−N-L−1. Lipid peroxidation (LPO), as indicated by the thiobarbituric acid reactive substances (TBARS) level, was significantly higher in fish exposed to 330 mg NO2−N-L−1 than the control and 20 mg NO2−N-L−1. Moreover, a pro-oxidant condition was induced when P. orbignyanus were exposed to concentrations from 80 mg NO2−N-L−1, which was evidenced by alterations in the antioxidant system and increased levels of LPO. Therefore, it is recommended that the nitrite concentration do not exceed 40 mg NO2−N-L−1 in order to avoid oxidative stress for early juvenile Brazilian flounder.