Shikonin is an anti‐inflammatory agent extracted from natural herbs. The aim of this study is to explain the treatment effects and mechanism of Shikonin in acute lung injury induced by sepsis. In this study, first, we evaluate different Shikonin concentrations for the anti‐inflammation of acute lung injury induced by sepsis in an in vivo study. On the basis of the results, we confirm that 50.0 mg/kg was the best therapeutic Shikonin concentration. As a second step, we discuss the mechanism of Shikonin by a vitro cell experiment. Finaly, we validate that Shikonin has effective treatment effects on acute lung injury via regulation of microRNA‐140‐5p/toll‐like receptor 4 (miRNA‐140‐5p/TLR4) in the in vivo study. The results of vitro and vivo study showed that Shikonin could improve acute lung injury induced by sepsis. The mechanism might be correlation miRNA‐140‐5p expression increasing, and regulated targeted gene TLR4, with TLR4 expression depressing, the downstream myeloid differentiation protein 88 and nuclear factor κB proteins expression were suppressed. In conclusion, Shikonin improved sepsis induced lung injury by regulation miRNA‐140‐5p/TLR4.