Objective To investigate the effect of the JAK2/ STAT3 signaling pathway on the Th17/ Treg imbalance in acute lung injury(ALI)and its mechanism. Methods Twenty-four C57BL/6 mice were randomly divided into control, model, JAK2 inhibitor, and STAT3 inhibitor groups with six mice in each group. The control group was instilled with normal saline in their airway, and the same amount of normal saline was injected intraperitoneally 8 h later. The model, JAK2 inhibitor, and STAT3 inhibitor groups were instilled with lipopolysaccharide(LPS) in their airway to establish the ALI model, and the same amount of normal saline, Fedratinib, and NSC74859 were injected intraperitoneally 8 hours later, respectively. The wet/ dry(W/ D) lung weight of mice was measured at 24 h after LPS infusion, the pathological changes of lung tissue were observed by HE staining, the proportion of Treg and Th17 cells in lung tissue was measured by flow cytometry, and IL-6, LL-10, IL-17A, and TGF-β1 levels in lung tissue homogenates were measured by ELISA. Protein expression of JAK2, STAT3, p-JAK2, and p-STAT3 in lung tissues was detected by Western blot. Results Compared with the control group, the W/ D ratio was increased(P<0. 01), lung tissue injury was severe, there was a degree number of inflammatory cell infiltration, the Th17/ Treg ratio, the levels of inflammatory factors IL-6, IL-17A, TGF-β1, p-JAK2, and p-STAT3 were significantly increased(P<0. 01), and the IL-10 level was significantly decreased in the model group(P<0. 01). Compared with the model group, the W/ D lung weight of JAK2 inhibitor and STAT3 inhibitor groups was decreased (P<0. 05), lung tissue injury was alleviated, inflammatory cell infiltration was reduced, the Th17/Treg ratio, IL-6, IL-17A, and TGF-β1 levels, p-JAK2 and p-STAT3 protein expression levels were significantly decreased in lung tissue(P<0. 01), and the IL-10 level was significantly increased(P<0. 01). Conclusions Inhibiting activation of the JAK2/ STAT3 signaling pathway regulates the imbalance of Th17/ Treg cells in lung tissue of ALI mice induced by LPS, inhibits the inflammatory response, and reduces lung injury.