Objective To investigate the expression of human follistatin-like protein 3 (FSTL3) in acute lung injury caused by different inducement in mice and its significance. Methods Mouse model of septic lung injury was established by intraperitoneal injection of lipopolysaccharide (LPS), and the model of ischemia-reperfusion (IR) injury was established by clamping the left pulmonary hilum followed by opening. The protein expression level of FSTL3 in serum and the protein and mRNA expression level of FSTL3 in lung tissue were detected at different time points of lung injury. FSTL3 neutralizing antibodies (FSTL3-NA) or FSTL3 gene knockout mice were employed to evaluate the effect of FSTL3 inhibition on lung injury in mice inflicted by 2 types of injuries. Western blotting was applied to detect the protein levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), superoxide dismutase (SOD) 1 and SOD2 in the lung tissues. Statistical software GraphPad Prism 9.2 was used to analyze the data. One-way AVOVA was used to compare the measurement data between groups, and student′s t test was performed for intergroup comparison. Results The serum protein level of FSTL3 was increased significantly in the mice at 12 h after intraperitoneal injection of LPS, reached a peak at 18 h, and then stabilized, both of which were significantly higher than 0 h (P<0.05). After 2 h ischemia and 2 h reperfusion, the serum protein level of FSTL3 was elevated significantly, summited at 6 h of reperfusion, and the protein level after 8 h reperfusion was still notably higher than that at 0 h (P<0.05). Western blotting and RT-PCR showed that the protein and mRNA levels of FSTL3 in the lung tissues reached the peaks after 18 h of LPS stimulation (P<0.05); after 2 h of ischemia and 2 h of reperfusion, its protein and mRNA levels in lung tissue were increased significantly (P<0.05), and reached the peaks after 4 h reperfusion (P<0.05). FSTL3-NA treatment obviously alleviated the lung pathological injury in sepsis mice, and down-regulated the protein levels of TNF-α[(1.23±0.11) vs (2.45±0.27)]and MCP-1 [(0.34±0.02)] vs (2.23±0.04)] in the lung tissue, while up-regulated the protein levels of SOD1 [(3.05±0.16) vs (1.87±0.32)] and SOD2 [(2.45±0.19) vs (1.61±0.24)] (all P<0.05). FSTL3 gene knockout also obviously alleviated the lung pathological injury in IR injured mice, and down-regulated the protein levels of TNF-α [(1.22±0.19) vs (4.13±0.21)] and MCP-1 [(0.88±0.03) vs (3.10±0.15)] in the lung tissue and up-regulated those of SOD1 [(2.27±0.16) vs (0.41±0.06)] and SOD2 [(2.04±0.13) vs (0.19±0.05)] (all P<0.05). Conclusion After LPS or IR stimulation, FSTL3 level is significantly increased in both mouse lung tissue and serum, while its inhibition or knockout could significantly reduce inflammation and oxidative stress in murine lung tissue.