Objective To investigate the role and mechanism of neutrophil extracellular traps (NETs) in acute liver injury from heatstroke (HS) mouse model. Methods Fifty-six C57BL/6 mice were randomly (random number table) assigned into sham heated control group (control group, n=8), sham heated with Chlorine amidine (CA) treatment group (CA group, n=8), heatstroke group (HS, n=32), and heatstroke with CA treatment group (HS+CA, n=8). In the HS group, after heat-shock treatment, mice were further subgrouped into HS 0 h, HS 3 h, HS 6 h, and HS 9 h (n=8/subgroup). Another forty-eight mice were randomized into the control group, CA group, HS(9 h) group, and HS+CA group (n=12/group). We tracked the survival rate of these mice up to 216 h. Mice were prepared with the pre-warm chamber to initiate HS. The change in rectum temperature (Tr) was monitored and the time point reaching 42.9 ℃ was recorded. At the end of heat stress, the mice were sacrificed according to the group time point, and the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured by the automatic biochemical analyzer. HE staining was used to observe the pathological injury of liver tissue. Plasma-free DNA and myeloperoxidase (MPO)-DNA complex concentrations were detected by the kit. The expression of citrullinated histone (Cit H3) and MPO in liver tissue was observed by a laser confocal microscope. The concentration of IL-1β in liver tissue homogenate was determined by ELISA.The activation level of Nlrp3 inflammasome in liver tissue was detected by Western blotting. Results There was no significant difference in heat exposure time and core body temperature rise rate between HS+CA group and HS group at the end of heat shock(P>0.05), but the survival time of HS+CA group was longer than that of HS group (P<0.05). At 3 h, 6 h, and 9 h after heat shock, the activities of serum ALT and AST, and the pathological scores of liver tissue increased progressively with time and were significantly higher than those in control group (P<0.001). At 3 h, 6 h, and 9 h after heat shock, the concentrations of plasma-free DNA and MPO-DNA, the levels of Cit H3 and MPO in liver tissue increased progressively with time and were significantly higher than those in control group (P<0.001). HE staining of liver tissue showed that, compared with HS group, the degeneration of hepatocytes in HS+CA group was significantly alleviated, inflammation was significantly alleviated, and there was little blood stasis in hepatic sinuses without bleeding. Inhibition of NETs release significantly alleviated heat stress-induced liver injury (HS+CA group vs. HS 9 h group, P<0.001). Survival analysis showed that the survival rate of mice in HS+CA group was higher than that in HS group (χ²=4.719,P<0.05). The expression levels of Nlrp3 and cleaved caspase-1 and IL-1β in liver tissue of HS+CA group were lower than those in HS group (P<0.001). Conclusion NETs may play an important role in the pathogenesis of HS liver injury through the Nlrp3 inflammasome/IL-1β signal pathway.