Chemokine CXC motif ligand 16 (CXCL16) is a multifaceted chemokine that has been shown to participate in a variety of inflammatory diseases. The role of CXCL16 in the immunopathology of sepsis remains unidentified. In this study, human patients with sepsis and healthy controls were used to obtain blood for in vitro studies, and female C57BL/6J mice were taken for in vivo studies. The effects of recombinant CXCL16 protein or anti-CXCL16 monoclonal antibody on sepsis were evaluated in a murine model of cecal ligation and puncture (CLP)–induced polymicrobial sepsis. On admission, human patients with sepsis had significantly higher soluble levels of serum CXCL16 than healthy controls. Soluble CXCL16 remained significantly elevated in septic patients from day 0 to 7. Admission levels of soluble CXCL16 were positively correlated disease severity and the serum levels of other inflammatory cytokines and chemokines. Furthermore, nonsurvivors displayed significantly higher admission levels of soluble CXCL16 compared with survivors of septic patients. Soluble CXCL16 levels revealed significant prognostic value for 28-day mortality, and CXCL16 was found to be an independent predictor of 28-day mortality in septic patients. In CLP-induced nonsevere sepsis, administration with recombinant CXCL16 increased mortality and tissue injury. Conversely, neutralizing CXCL16 by anti-CXCL16 monoclonal antibody decreased mortality and tissue injury in CLP-induced severe sepsis. However, CXCL16 did not affect the ability of these mice to clear bacteria in CLP. Taken together, CXCL16 could be linked to sepsis not only as a new marker of prognosis, but also as a potential target for therapeutic intervention.