ABSTRACT: Purpose: We previously identified interleukin 27 (IL-27) as a sepsis diagnostic biomarker in critically ill children. The current study tested the performance of IL-27 alone and in combination with procalcitonin (PCT) for diagnosing sepsis in critically ill adults. Methods: Serum samples were made available from a prior prospective study of sepsis biomarkers in critically ill adults. The primary analysis used receiver operating characteristic curves to evaluate the performance of IL-27 and PCT. Secondary analysis explored IL-27 performance in subgroups of patients with sepsis secondary to lung and nonlung sources of infection. The net reclassification improvement was used to estimate the incremental predictive ability of IL-27 compared with PCT alone. Classification and regression tree analysis was used to generate an IL-27- and PCT-based decision tree. Results: There were 145 patients with sepsis and 125 without sepsis. The receiver operating characteristic curve for IL-27 was inferior (area under the curve [AUC], 0.68; 95% confidence interval [CI], 0.62–0.75) to that of PCT (AUC, 0.84; 95% CI, 0.79–0.89). Similar findings were observed when comparing patients with a lung source of infection and those without sepsis. For sepsis patients with a nonlung source of infection, adding IL-27 to PCT improved discrimination (net reclassification improvement = 0.685; P < 0.001). The AUC for the classification and regression tree–derived decision tree was 0.92 (95% CI, 0.88–0.96) and was significantly greater than that of PCT alone. Conclusions: When used in combination with PCT, IL-27 may improve classification of critically ill adults with sepsis secondary to a nonlung source of infection.