Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern. Neutrophils present in the mononuclear cell fraction of Ficoll gradient separation are called low-density neutrophils (LDNs). Here we report the novel role of eCIRP on LDNs’ heterogeneity in sepsis. Sepsis was induced in male C57BL/6 wild-type (WT) and CIRP^−/− mice by cecal ligation and puncture (CLP). At 20 h after CLP, LDNs in the blood were isolated by Ficoll gradient separation, followed by staining the cells with anti-Ly6G and anti-CD11b Abs and detection by flow cytometry. Sepsis or recombinant murine CIRP (rmCIRP) injection in mice resulted in significant increase in the frequency (%) and number of Ly6G⁺CD11b^hi and Ly6G⁺CD11b^lo LDNs in the blood compared to sham- or vehicle-treated mice. At 20 h of CLP, CIRP^−/− mice had significantly lower frequency and number of Ly6G⁺CD11b^hi and Ly6G⁺CD11b^lo LDNs in the blood compared to WT mice. In sepsis mice or rmCIRP-injected mice, compared to Ly6G⁺CD11b^lo LDNs, the expression of CXCR4, ICAM-1, and iNOS and formation of reactive oxygen species, and neutrophil extracellular traps in Ly6G⁺CD11b^hi LDNs in the blood were significantly increased. Treatment of WT bone marrow-derived neutrophils (BMDNs) with rmCIRP increased Ly6G⁺CD11b^hi LDN frequency, whereas treatment of TLR4^−/− BMDNs with rmCIRP significantly decreased the frequency of Ly6G⁺CD11b^hi LDNs. BMDNs’ stimulation with rmCIRP increased the expression of transcription factors in LDNs. eCIRP induces the formation of a proinflammatory phenotype Ly6G⁺CD11b^hi of LDNs through TLR4. Targeting eCIRP may provide beneficial outcomes in sepsis by decreasing proinflammatory Ly6G⁺CD11b^hi LDNs.