In vitro, RAW 264.7 cells were pretreated with different dose of Isotetrandrine 1 h before treatment with 1 mg/L of LPS. In vivo, to induce ALI, male BALB/c mice were injected intranasally with LPS and treated with Isotetrandrine (20 and 40 mg/kg) 1 h before LPS. In vitro, the cytokine levels of tumor necrosis factor-α±, interleukin (IL)-1β, and IL-6 in supernatant were reduced by Isotetrandrine. Meanwhile, in vivo, pulmonary inflammatory cell infiltration, myeloperoxidase activity, total cells, neutrophils, macrophages, along with the levels of tumor necrosis factor-α±, IL-1β, and IL-6 in bronchoalveolar lavage fluid were dose-dependently attenuated by Isotetrandrine. Furthermore, our data showed that Isotetrandrine significantly inhibited the activation of MAPK and NF-κB, which are induced by LPS in ALI model.