Objective To investigate the regulatory role of programmed death ligand-1 (PD-L1) on acute lung injury (ALI), and its molecular mechanism. Methods Twenty C57BL/6 male mice and 20 PD-L1 knock out male mice were collected, and they were divided into two groups by random number table, respectively: namely sham group and ALI group, 10 mice in each group. The model of ALI was reproduced by two-hit of hemorrhagic shock and sepsis, and the mice in sham group were only got bilateral femoral artery exposure and ligation without bleeding, cecal separation without ligation and perforation. The mice were sacrificed 24 hours after model reproduction, and the lung tissue and bronchoalveolar lavage fluid (BALF) were collected. The mRNA and protein expression levels of PD-L1 in the lungs were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot. The pathological changes were observed with microscopy. The protein levels in BALF were determined. The granulocyte differentiation antigen 1 (Gr1) positive cells was determined by cytometry, and myeloperoxidase (MPO) activity in lung tissue was determined. The levels of proinflammatory factors interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and chemotatic factors keratinocyte chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2) in lung homogenates and BALF were determined by enzyme-linked immunosorbent assay (ELISA). Results Compared with sham group, the mRNA and protein levels of PD-L1 in lung tissue of C57BL/6 mice in ALI group were significantly elevated [PD-L1 mRNA (2-ΔΔCt): 3.20±0.76 vs. 1.01±0.03, PD-L1 protein (A value): 0.98±0.16 vs. 0.15±0.04, both P < 0.05]. It was shown by light microscopy that the alveolar wall was thickened, congestive, edema and spot bleeding with a large number of inflammatory cell infiltration in the lung tissue of C57BL/6 mice in ALI group, and an obvious protein leakage was found in BALF (ng/L: 0.18±0.06 vs. 0.05±0.01, P < 0.05). The lung injury degree of PD-L1 knockout ALI mice was significantly less than that of C57BL/6 ALI mice, and the protein leakage was significantly reduced in BALF (ng/L: 0.11±0.02 vs. 0.18±0.06, P < 0.05). Compared with corresponding sham group, the number of Gr1 positive cells, MPO activity in lung tissue as well as the levels proinflammatory factors and chemotatic factors in lung tissue and BALF in ALI group were significantly increased. However, when compared with C57BL/6 ALI mice, above parameters in lung homogenates and BALF were significantly decreased in PD-L1 knockout ALI mice [number of Gr1 positive cells: (39.0±4.0)% vs. (45.0±8.0)%, MPO activity (U·μg-1·min-1): 2.85±0.62 vs. 4.52±1.16; lung IL-6 (ng/g): 461±111 vs. 728±28, TNF-α (ng/g): 1 123±175 vs. 1 500±327, KC (ng/g): 150±34 vs. 250±28, MIP-2 (ng/g): 1 263±468 vs. 1 763±323; BALF IL-6 (ng/L): 134±22 vs. 258±38, TNF-α (ng/L): 598±102 vs. 889±139, KC (ng/L): 934±286 vs. 1 258±336, MIP-2 (ng/L): 650±130 vs. 950±256; all P < 0.05]. Conclusion PD-L1 may play an important protective role in the immunological mechanism of ALI, which may be mediated by decreasing chemotactic factor KC and MIP-2 and mitigating neutrophil chemotaxis in lung tissue.