Rationale: Costimulatory molecules, including the CD40-CD154 and CD80/86- CD28 dyads play a prominent role in regulating inflammation in the adaptive immune response. Recent studies from our group and others suggest a potentially important role for these costimulatory cascades in innate immunity as well. Objectives: To determine the role of CD80/86 alone and in combination with CD40 in lethal polymicrobial sepsis in mice and humans. Methods: The murine Cecal Ligation and Puncture (CLP) model was used to determine the role of CD80/86 alone and in combination with CD40 using WT, CD80/86^-/- mice and a novel CD40/80/86^-/- mouse. Expression of cell bound and soluble costimulatory molecules were assessed in humans via ELISA and FACS. Measurements and Results: Lethal CLP was associated with upregulation of CD40 and CD80/86 and their respective ligands CD28 and CD154 on innate effector cells. Blockade or deletion of CD80/86 attenuated mortality and inflammatory cytokine production during CLP. CD40/80/86^-/- mice exhibited further reductions in mortality, lung injury and inflammatory cytokine production compared to CD80/86^-/- mice. Finally, humans with sepsis had increased monocyte expression of CD40 and CD80 compared to healthy controls; with higher levels in subjects requiring vasopressor support. Levels of soluble CD28 and CD154 were significantly higher in patients who died compared to those who lived. Conclusions: These data demonstrate a central role for CD40 and CD80/86 in the innate immune response and suggest that combined inhibition of CD40 and CD80/86 may improve mortality in sepsis. Expression of costimulatory molecules may serve as biomarkers for outcome in septic patients.