Objective To investigate the role of autophagy in acute lung injury in rats with exertional heat stroke (EHS). Methods A total of 60 male Wistar rats were randomly divided into four groups: control group, EHS group, EHS+bafilomycin A1 (Baf A1) group, and EHS+rapamycin (RAPA) group, with 15 rats in each group. The EHS+Baf A1 group (Baf A1, 1 mg/kg)and EHS+RAPA group (RAPA, 1 mg/kg) were injected intraperitoneally for 4 consecutive days before modeling the rats. After establishing the EHS model, we observed for 5 hours, followed by anesthesia for these rats to collect tissues. We observed and compared the morphological changes in lung tissue, performed arterial blood gas analysis, and measured the lung coefficient and Evans blue (EB) leakage. We also examined the lung tissue pathological changes using HE staining, evaluated lung tissue apoptosis through the TdT-mediated dUTP Nick-end labeling (TUNEL) method, and detected microtubule-associated protein Ⅰ light chain 3(LC3)-Ⅱ/LC3-Ⅰ ratio in lung tissue and p62 protein level through Western blotting. Results Compared with control group,the lung tissue of the EHS group was obviously swollen and more fluid exuded; the arterial blood oxygen partial pressure (PaO₂)decreased, while the arterial blood carbon dioxide partial pressure (PaCO₂) increased; the lung coefficient and EB leakage were significantly increased; HE staining and apoptosis staining showed uneven thickening of alveolar septum and alveolar wall, alveolar exudation increased and the number of apoptotic cells increased, the difference was statistically significant (P<0.05); Compared with EHS group, the EHS+Baf A1 group had more severe lung tissue swelling, lung coefficient and EB leakage were increased; PaO₂ further decreased, while PaCO₂ further increased; HE staining and apoptosis staining showed that lung tissue lesions and damage were significantly worsened, and the number of apoptotic cells increased significantly (P<0.05). Compared with EHS group, the EHS+RAPA group had a significant decrease in various pulmonary edema indicators; PaO₂ increased, PaCO₂ is close to the normal range; HE staining and apoptosis staining showed lung tissue lesions and the degree of damage was significantly reduced, and the number of apoptotic cells was reduced, and the difference was statistically significant (P<0.05); Western blotting results showed that compared with control group, the ratio of LC3-Ⅱ/LC3-Ⅰ in the lung tissue of rats in the EHS group was significantly reduced(0.3±0.1 vs. 1.0±0.1, P<0.05), the expression of p62 increased (1.4±0.2 vs. 0.8±0.1, P<0.05). Compared with EHS group, the ratio of LC3-Ⅱ/LC3-Ⅰ in the lung tissue of the EHS+Baf A1 group was reduced (0.10±0.04), and the expression of p62 was further increased (1.7±0.1), while the ratio of LC3-Ⅱ/LC3-Ⅰ in the lung tissues of rats in the EHS+RAPA group was significantly increased (0.5±0.1), and the expression of p62 was significantly decreased (1.1±0.1), the differences were statistically significant(P<0.05). Conclusion The reduction of autophagy levels is one of the mechanisms of EHS leading to lung injury. Up-regulation of autophagy can reduce the acute lung injury of EHS.