Background: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are serious clinical disease entities characterized by inflammatory pulmonary edema, which lead to acute hypoxic respiratory failure through various etiologies. According to the studies to date, ALI/ARDS has been recognized as a form of multiorgan failure related to overactive immune response, and overproduction of proinflammatory cytokines released from activated inflammatory cells are considered to play a key role in the development of ALI. Glycyrrhizin (GL) is an extractive component derived from Glycyrrhiza glabra (licorice), which has recently been reported to have various pharmacological effects like anti-inflammatory, anti-tumor, hepatoprotective, and anti-viral activities. Nevertheless, the therapeutic effect of GL in ALI is still unclear. The aim of this study was to investigate therapeutic effects of GL on lipopolysaccharide (LPS)-induced ALI in a mouse model and to elucidate explicable mechanisms involved. Methods: A total of 36 BALB/c mice (6-week-old, 27.7±1.9-gram body weight) were randomly divided into 3 groups: the control group (normal saline was administered intravenously, n=10), the LPS group (LPS 50 mg/kg was intraperitoneally administered, n=13), and the LPS + GL group (GL was administered intravenously immediately and 12 hours after LPS injection, n=13). Mice were sacrificed after 24 hours, and bronchoalveolar lavage fluid (BALF) was collected for the estimation of protein content, inflammatory cell counts, proinflammatory cytokines, myeloperoxidase (MPO) activity, and expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB). Then, the lungs were excised for molecular target, histopathological, and immunohistochemical examinations. Results: Compared to the LPS group, GL significantly decreased protein content, inflammatory cell counts, tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-6, MPO activity, and expressions of COX-2, iNOS, and NF-κB in the LPS + GL group. GL attenuated migration and infiltration of inflammatory cells, showing a marked decrease in CD 11b-positive cells (26.77%±0.83% vs. 41.77%±0.81% vs. 23.23%±1.92%, P<0.05) as well as CXCR4−/CXCR1-positive cells (CXCR4: 37.23%±1.00% vs. 59.37%±2.37% vs. 47.45%±4.36%; CXCR1: 32.10%±1.56% vs. 47.03%±1.99% vs. 21.70%±6.50%; all P<0.05) in the control, LPS, and LPS + GL groups. Additionally, immunohistochemistry showed that the expression of Toll-like receptor 4 (TLR-4) was inhibited by GL. Conclusions: The results of this study indicate that GL may have anti-inflammatory and protective effects on LPS-induced ALI in mice. GL inhibited proinflammatory cytokines playing a key role in the initial phase of inflammatory response, which suggests that inhibition of the TLR-4/NF-κB signal pathway would be a possible mechanism underlying the action of GL. Thus, GL can be used as a novel therapeutic strategy for pulmonary inflammation.