Recent data suggest that short-chain fatty acids (SCFAs), the major fermentation product from gut microbial degradation of dietary fiber, have protective effects against renal ischemia-reperfusion (IR) injury, colitis, and allergic asthma. However, the effect of SCFAs on acute lung injury (ALI) caused by IR is still unclear. In this study, we examine whether SCFAs have protective effects against IR-induced ALI and explore possible protective mechanisms. IR-induced ALI was established by 40 min ischemia followed by 60 min reperfusion in isolated perfused rat lungs. Rats were randomly assigned to one of six groups: control, control + acetate (400 mg/kg), IR, and IR + acetate at one of three dosages (100, 200, 400 mg/kg). Bronchoalveolar lavage fluids (BALF) and lung tissues were obtained and analyzed at the end of the experiment. In vitro, mouse lung epithelial cells (MLE-12) subjected to hypoxia-reoxygenation (HR) were pretreated with acetate (25 mmol/L) and GPR41 or GPR43 siRNA. Acetate decreased lung weight gain, lung weight/body weight ratios, wet/dry weight ratios, pulmonary artery pressure, and protein concentration of the BALF in a dose-dependent manner for IR-induced ALI. Acetate also significantly inhibited the production of TNF-α, IL-6 and CINC-1 in the BALF. Moreover, acetate treatment restored suppressed IκB-α levels and reduced nuclear NF-κB p65 levels in lung tissues. In addition, acetate mitigated IR-induced apoptosis and tight junction disruption in injured lung tissue. In vitro analyses showed that acetate attenuated NF-κB activation and KC/CXCL-1 levels in MLE-12 cells exposed to HR. The protective effects of acetate in vitro were significantly abrogated by GPR41 or GPR43 siRNA. Acetate ameliorates IR-induced acute lung inflammation and its protective mechanism appears to be via the GPR41/43 signaling pathway. Based on our findings, acetate may provide a novel adjuvant therapeutic approach for IR-induced lung injury.