Neutrophils are critically involved in host defense and tissue damage. Intrinsic signal mechanisms controlling neutrophil activities are poorly defined. We found that the expression of wild-type p53-induced phosphatase 1 (Wip1) in mouse and human neutrophils was downregulated quickly after neutrophil activation through JNK-microRNA-16 pathway. Importantly, the Wip1 expression level was negatively correlated with inflammatory cytokine productions of neutrophils in sepsis patients. Wip1-deficient mice displayed increased bactericidal activities to Staphylococcus aureus and were hypersensitive to LPS-induced acute lung damage with increased neutrophil infiltration and inflammation. Mechanism studies showed that the enhanced inflammatory activity of neutrophils caused by Wip1 deficiency was mediated by p38 MAPK-STAT1 and NF-κB pathways. The increased migration ability of Wip1KO neutrophils was mediated by the decreased CXCR2 internalization and desensitization, which was directly regulated by p38 MAPK activity. Thus, our findings identify a previously unrecognized function of Wip1 as an intrinsic negative regulator for neutrophil proinflammatory cytokine production and migration through multiple signal pathways.