Objective To investigate the effect of total flavonoids of selaginella officinalis (TFST) regulating the Nrf2/HO-1 signaling pathway on mechanical ventilation associated lung injury (VALI). Methods Forty healthy SPF grade male SD rats were selected and randomly divided into four groups (10 in each group) using a random number table method. The control group only received intubation without mechanical ventilation; In the model group, a rat VALI model was prepared using tidal volume of 30 mL/kg, ventilation frequency of 60 times/min, oxygen uptake concentration of 21% FiO2, inhalation to exhalation ratio of 1∶2, and ventilation time of 4 hours; TFST group, starting from 2 weeks before modeling, was orally administered 800 mg/kg of total flavonoids suspension of selaginella officinalis once a day; ML385 group, pre protected with total flavonoids of selaginella vulgaris for 2 weeks, and ML385 (Nrf2 inhibitor) 30mg/kg was injected into the intraperitoneal cavity of rats 1 hour before mechanical ventilation. After the end of the experiment, the rats were euthanized and their bronchoalveolar lavage fluid (BALF) was collected for the determination of IL-6 and TNF- α using ELISA concentration. And collect rat lung tissue for HE staining to observe its pathological changes, and then score lung injury. Compared the wet to dry weight ratio (W/D) of lung tissue in each group of rats, detected the level of malondialdehyde (MDA) in lung tissue, measured the level of superoxide dismutase (SOD) in lung tissue using WST-8 method, and detected the levels of Nrf2 and HO-1 protein in lung tissue using Western blot method. Results Compared with the control group, the W/D value and MDA content of lung tissue, lung injury score, and IL-6 and TNF-α in BALF in the model group were elevated. SOD content, Nrf2 and HO-1 protein levels in lung tissue decreased (P＜0.05). Compared with the model group, the W/D value and MDA content of lung tissue in the TFST group decreased, lung injury decreased, and IL-6 and TNF- α in BALF decreased decreased. SOD content, Nrf2, HO-1 protein levels increased (P＜0.05). The ML385 group had more severe lung injury compared to the TFST group, with W/D values and MDA content in lung tissue, as well as IL-6 and TNF- α in BALF elevated, SOD content and Nrf2 and HO-1 protein levels decreased (P＜0.05). Conclusion The total flavonoids of selaginella can improve mechanical ventilation related lung injury in rats by regulating the Nrf2/HO-1 pathway and activating antioxidant pathways