Objective To investigate the effect of Sirtuin3 (Sirt3) on pulmonary epithelial apoptosis in severe acute pancreatitis (SAP) induced acute lung injury (ALI) and the underlying molecular mechanism. Methods Among 35 wild‑type mice and 35 Sirt3 knockout mice, and 34 mice each were intraperitoneally injected with caerulein (50 μg/kg) and lipopolysaccharide (LPS, 10 mg/kg) to construct a SAP‑ALI model. The survival rate was analyzed at 12, 24, 36, 48, 72 h after injection. The inflammatory condition of lung tissue in wild‑type mice and gene knockout mice was observed by hematoxylin‑eosin (H‑E) staining 12 h after injection. Before injection and at 8, 12, and 24 h after injection, protein levels of cysteinyl aspartate specific proteinase (caspases)‑3, caspase‑9 and X‑linked inhibitor of apoptosis‑associated factor (Xaf1) in the lung tissues of wild‑type mice and gene knockout mice were measured by Western blot. Another wild‑type mouse and gene knockout mouse were euthanized and their lung tissues were collected for measurement of the protein expression of Xaf1 by immunohistochemical staining. MLE12 cells were divided into four groups (1 well in each group): blank control group 1 (group A), tumor necrosis factor‑α (TNF‑α) stimulation at 20 μg/L for 36 h group (group B), Sirt3 interference+TNF‑α stimulation at 20 μg/L for 36 h group (group C), and Sirt3 overexpression+TNF‑α stimulation at 20 μg/L for 36 h group (group D). The apoptosis rates of four groups of cells were examined by flow cytometry. MLE12 cells were then divided into three groups (1 well in each group): blank control group 2 (group E) and Sirt3 interference+TNF‑α stimulation at 20 μg/L for 36 h group (group F), and Sirt3 and Xaf1 interference+TNF‑α stimulation at 20 μg/L for 36 h group (group G). The protein expression and enzyme activity of caspase‑3 and caspase‑9 after interfering Xaf1 were analyzed in groups E, F, and G. Results The survival rates of Sirt3 knockout mice 12, 24, 36, 48, 72 h after injection were lower than those of wild‑type mice (P<0.05). Compared with wild‑type mice, gene knockout mice showed more significant infiltration of inflammatory cells and interstitial edema in the lung tissues12 h after injection. Then, 8, 12, 24 h after injection, the levels of caspase‑3 and caspase‑9 in the lung tissues of gene knockout mice were higher than those of wild‑type mice (P<0.05), while the levels of Xaf1 was also higher than those of wild‑type mice (P<0.05). The apoptosis rates of group A, group B, group C, and group D were 11%, 21%, 33%, and 7% respectively. The levels of caspase‑3 and caspase‑9 in group F were higher than those in group E (P<0.05), while the activities of caspase‑3 and caspase‑9 was higher than those in group E (P<0.05). The amounts of caspase‑3 in group G were lower than those in group F (P<0.05), while the activities of caspase‑3 and caspase‑9 were lower than those in group F (P<0.05). Conclusions Sirt3 regulates the expression of Xaf1, inhibits the apoptosis of lung epithelial cells under SAP, and reduces the mortality rate of SAP‑ALI.