Acacetin, a natural product, has a wide spectrum of biological activities such as antioxidant properties. In the present study, we examined whether Acacetin has any beneficial role on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and, if so, whether its effect is mediated via heme oxygenase-1 (HO-1), an antioxidant enzyme playing an important role in ALI. Male BALB/c mice were stimulated with LPS intratracheal instillation to induce ALI. Acacetin was administrated 2h after LPS challenge. Samples were harvested 10h after LPS administration. We demonstrated that LPS challenge significantly induced lung histological alterations such as inflammation and edema. Acacetin administration notably attenuated these changes and reduced tumor necrosis factor-α and interleukin-1β in lung tissues. The LPS-induced reactive oxygen species generation was markedly suppressed by Acacetin. Furthermore, Acacetin treatment significantly elevated pulmonary HO-1 and nuclear factor erythroid-2-related factor 2 (Nrf2) activities. However, the beneficial action of Acacetin was markedly abolished when pretreated with zinc protoporphyrin, an inhibitor of HO-1. In in vitro studies, Acacetin notably increased the HO-1 expression in pulmonary microvascular endothelial cells. During knockdown of Nrf2 by siRNA, the effect of Acacetin on HO-1 expression was significantly reversed. Acacetin attenuates LPS-induced ALI in mice. This protective effect of Acacetin may be mediated, in part, through an HO-1-dependent pathway.