Neutrophils are abundant innate immune cells with important roles in host defense and many inflammatory diseases. They possess plasticity and heterogenicity, which underlie their functional ambivalency; they can be beneficial or detrimental to host well-being depending on contexts. While bone marrow is often regarded as the primary source of neutrophil production, the roles of extramedullary production in regulating neutrophil plasticity and heterogenicity in the context of autoimmune diseases remain poorly understood. Here, we report that the lack of both WNT5A and WNT5B unleashes anti-inflammatory protection in mice from Dextran Sodium Sulfate-induced colitis, accompanied with reduced colonic CD8+ T cell activation and enhanced splenic extramedullary myelopoiesis. By using the proteomics method to analyze the blood neutrophils from WT and Wnt5 DKO mice under DSS treatment, our analysis revealed elevated CD101 expression in blood neutrophils from Wnt5 DKO mice, leading to the suppression of CD8 T cell activation in vitro. This proteomic investigation unveiled the mechanism through which neutrophils from Wnt5 DKO mice inhibit CD8 T cells activation.