Mechanical ventilation has significant therapeutic benefits, but it may cause or aggravate lung injury, which is called ventilator-induced lung injury (VILI). Endogenous hydrogen sulfide (H2S) has roles including regulating inflammation, and promoting vasodilatation; it also exhibits anti-oxidative stress and anti-fibrosis effects. H2S has been reported to alleviate lung injury, but the effects and mechanism of H2S on VILI remain unclear. The present study established a rat model of VILI and treated them with H2S, then measured the changes in respiratory function indicators, lung tissue histopathology, and oxidative, inflammatory, and apoptotic indicators. The effect of H2S on autophagy in the VILI model and the involvement of endoplasmic reticulum (ER) stress were also investigated. To further explore the mechanism, L2 alveolar epithelial cells were treated with cyclic strain to mimic mechanical strain along with the H2S donor NaHS, and the involvement of the NF-κB/MAPK signaling pathway was examined. The results showed that H2S significantly alleviated VILI and inhibited the inflammation and oxidative stress induced by VILI. H2S also significantly reduced autophagy and ER stress in rats. The phosphorylation of IRE1α, PERK and eIF2α and the expression of nuclear ATF4, and GADD34 in L2 cells were all significantly reduced with NaHS. Nuclear NF-κB p65, MAPK p38, JNK, and ERK were all activated by cyclic strain, but inhibited by the ER stress inhibitor 4-PBA or NaHS. Our findings revealed that H2S treatment alleviated VILI by regulating autophagy and ER stress, and the PERK/eIF2α/ATF4/GADD34 and NF-κB/MAPK pathways were involved in the underlying mechanism.