The physiological indexes and intestinal microflora of Cherax quadricarinatus overwintering were studied at different concentrations of nitrite. The acute toxicity of nitrite to Cherax quadricarinatus was studied by conventional bioacute toxicity test method, and the semi-lethal concentration of 96 h was 22.0 mg/L and the safe concentration was 2.2 mg/L. Four groups of nitrite experimental groups were set up, respectively, group A (0 mg/L), group B (0.5 mg/L), group C (2.3 mg/L) and group D (5.0 mg/L).Samples were taken after 6 weeks of stress. The results showed that:There was no significant difference in survival rate, hypertrophy and liver index in four groups (P>0.05), but the survival rate, weight gain rate and body length increase rate of group D were the lowest. Hepatopancreas histology analysis:With the increase of nitrite concentration, the arrangement of hepatic tubules tended to be disordered and the sizes of hepatic tubules became different. The connective tissue between hepatic tubules decreased and vacuoled, especially in group D. Physiological biochemistry indicators analysis:SOD activity decreased with the increase of nitrite concentration. In hepatopancreas, group A was significantly higher than the other three groups (P<0.05), and group D was significantly lower than the other three groups (P<0.05). In muscle, group A was significantly higher than group C and D (P<0.05), and group D was significantly lower than group A and B (P<0.05). MDA activity increased with the increase of nitrite concentration. In hepatopancreas, there was no significant difference among the four groups (P>0.05). In muscle, group A was significantly lower than group C and D (P<0.05). Immune-related indicators decreased with the increase of nitrite concentration. In hepatopancreas, ACP and AKP activities of four groups were significantly different (P<0.05), the highest in group A and the lowest in group D. In muscle, ACP activity in group A was significantly higher than that in other three groups (P<0.05). AKP activity in four groups was significantly different (P<0.05), with the highest in group A and the lowest in group D. UL activity had no significant difference in each group (P>0.05). The activity of energy metabolic index TG decreased with the increase of nitrite concentration, and the TG activity in group A was significantly higher than that in group D (P<0.05). Intestinal flora analysis:Nitrite stress increased the proportion of Enterobacteriaceae to a certain extent, and the diversity of group D was the lowest and that of group A was the highest. Nitrite caused the decrease of intestinal flora diversity.