BACKGROUND Sepsis-induced lung injury is associated with high mortality. The present investigation evaluated the protective effect of hesperidin against sepsis-induced lung injury and also postulates the possible mechanism of its action. MATERIAL AND METHODS Lung injury was induced by sepsis in all animals, in which sepsis was produced by cecal ligation and puncture (CLP). Animals were treated with hesperidin 10 and 20 mg/kg i.v. 30 min after the surgery. Oxygenation index and lung injury score were determined and levels of pro-inflammatory mediators and markers of oxidative stress were also estimated in the lung tissues. Moreover, expression of caspase-3, B-cell lymphoma (Bcl-2), Toll-like receptor 4 (TLR4), heat-stable protein 70 (Hsp70) and myeloid differentiation primary response 88 (MyD88) protein was estimated by Western blot assay and immunofluorescence assay. RESULTS Hesperidin attenuated the partial pressure of arterial oxygen/fraction of inspired oxygen (PaO2/FiO2) ratio and lung injury score in CLP-induced lung injury mice. There was a significant (p<0.01) decrease in the level of pro-inflammatory mediators in the lung tissue of CLP-induced lung injury mice. Moreover, markers of oxidative stress were attenuated in the hesperidin-treated group. Treatment with hesperidin attenuated the expression of caspase-3, Bcl-2, TLR4, Hsp70, and MyD88 protein in the lung tissue of CLP-induced lung injury mice. CONCLUSIONS Hesperidin protects against lung injury by attenuating the Hsp70/TLR4/MyD88 pathway in CLP-induced lung injury mice.