Acute lung injury (ALI) is a common acute and severe disease in clinical practice. Staphylococcal EnterotoxinB (SEB) is a superantigen that can cause inflammatory ALI. MiR-222 has been demonstrated to be upregulatedin SEB-induced inflammatory ALI, but its exact roles and functions remain ill-defined. In this study, SEBexposure led to inflammatory ALI and high expression of miR-222 in model mice and lung infiltratingmononuclear cells, but the inflammatory response and high expression of miR-222 were restored in miR-222-/-mice. Moreover, we investigated the roles of miR-222 in vitro and observed that the concentrations ofinflammatory cytokines and the expression of miR-222 were all elevated in SEB-activated splenocytes andmiR-222 inhibition reversed the effects. Foxo3 was confirmed as a direct target of miR-222. Interestingly, SEBexposure led to a decrease of Foxo3 expression, and Foxo3 knockdown partially reversed the promotion ofFoxo3 and the inhibition of inflammatory cytokines induced by miR-222 inhibitor in SEB-activated splenocytes.Our data indicated that miR-222 inhibition could alleviate SEB-induced inflammatory ALI by directlytargeting Foxo3, shedding light on the potential therapeutic of miR-222 for SEB-induced inflammation in thelung.