Abstract: 【Objective】 The aim of this study was to investigate the effects of combined stress of high temperature and nitrite on osmoregulation related indexes of Litopenaeus vannamei, which could provide theoretical basis for the healthy culture of Litopenaeus vannamei. 【Method】 Two temperature treatments were set up in the experiment:normal temperature group（25 ℃） and high temperature group（34 ℃）, and six nitrite concentration treatments were set up under each temperature condition:0.05（control）, 0.2, 0.8, 3.2, 6.4, and 12.8 mg/L. The water temperature was adjusted and maintained stable by a heating rod, and nitrite was used to regulate the nitrite concentration in the water. Samples were taken at 0, 6, 72 and 168 h, and gill tissues were dissected on ice. Hemolymph was extracted from the pericardial cavity with a 1.0 mL sterile syringe and mixed with anticoagulant. The absorbance at 340 nm was measured using an ultraviolet spectrophotometer, and the hemocyanin concentration in the hemolymph was calculated. The hemolymph osmotic pressure of Litopenaeus vannamei was measured using an osmometer. Na⁺/K⁺-ATP andCa²⁺/Mg²⁺-ATPase activity assay kits were used to determine the activity of Na⁺/K⁺-ATP and Ca²⁺/Mg²⁺-ATPase. Real-time fluorescence quantitative PCR was used to detect the relative expression of osmotic pressure regulation related genes in the gill tissues of Litopenaeus vannamei. 【Result】 The results of hemocyanin concentration test showed that at 25 ℃ water temperature, the hemocyanin concentrations of 3.2, 6.4, and 12.8 mg/L nitrite groups were significantly lower than those of 0.05, 0.2, and 0.8 mg/L nitrite groups after nitrite stress for 6, 72 and 168 h（P＜0.05, the same below）. At 25 and 34 ℃ water temperatures, the hemocyanin concentration of 12.8 mg/L nitrite group was the lowest after nitrite stress for 168 h. The results of osmotic pressure measurement showed that at 25 and 34 ℃ water temperatures, the hemolymph osmotic pressure of the 6.4 and 12.8 mg/L nitrite groups decreased with the increase of stress time. The results of enzyme activity detection showed that at 34 ℃ water temperature, after nitrite stress for 6 h, the Ca²⁺/Mg²⁺-ATPase activity in the gill tissue of the 6.4 mg/L nitrite group was the lowest, and at 25 and 34 ℃ water temperatures, after nitrite stress for 72 and 168 h, the Na⁺/K⁺-ATPase activity in the gill tissue of the 12.8 mg/L nitrite group was the lowest. The results of real-time fluorescence quantitative PCR showed that at 25 ℃, the relative expression of CLC gene increased gradually with the increase of nitrite concentration after nitrite stress for 6 h, and the relative expression of CCP gene was the highest in the 12.8 mg/L nitrite concentration group. 【Conclusion】 The combined stress of high temperature and nitrite affects the hemocyanin concentration and hemolymph osmotic pressure of Litopenaeus vannamei, leading to changes in the activity of osmoregulation related enzymes and relative expression of genes in the gill tissue of Litopenaeus vannamei. Nitrite stress can cause physiological disorders in the gill tissue of Litopenaeus vannamei, and high temperature can aggravate the toxicity of nitrite and affect the physiological function of the gill tissue of Litopenaeus vannamei.