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Background: Multiple organ dysfunction is a complex and lethal clinical feature with heterogeneous causes and is usually characterized by tissue injury of multiple organs. Tenascin-C (TNC) is a matricellular protein that is rarely expressed in most of the adult tissues, but re-induced following injury. This study aimed to evaluate serum TNC in predicting mortality in critically ill patients with multiple organ dysfunction.Methods: Adult critically ill patients with at least two organs dysfunction and an increase of Sequential Organ Failure Assess (SOFA) score ≥ 2 points within 7 days were prospectively enrolled into two independent cohorts. The emergency (derivation) cohort was a consecutive series and the patients were from Emergency Department. The inpatient (validation) cohort was a convenience series and the patients were from medical wards. Their serum samples at the first 24 h after enrollment were collected and subjected to TNC measurement using ELISA. The association between serum TNC level and 28-day all-cause mortality was investigated, and then the predictive value of serum TNC was analyzed.Results: A total of 110 patients with a median age of 64 years (53, 73) were enrolled in the emergency cohort. Compared to the survivors, serum TNC in the non-survivors was significantly higher (467.7 vs. 197.5 ng/ml, p < 0.001). Multivariate logistic regression analysis revealed that the association between serum TNC and 28-day mortality was independent of sepsis or critical illness scores such as SOFA, Acute Physiology and Chronic Health Evaluation (APACHE II), and Simplified Acute Physiology Score (SAPS II), respectively (p < 0.001 for each). The area under receiver operating characteristic curve of serum TNC for predicting mortality was 0.803 (0.717–0.888) (p < 0.001), similar with SOFA 0.808 (0.725–0.891), APACHE II 0.762 (0.667–0.857), and SAPS II 0.779 (0.685–0.872). The optimal cut-off value of serum TNC was 298.2 ng/ml. Kaplan–Meier analysis showed that the survival of patients with serum TNC ≥ 300 ng/ml was significantly worse than that of patients with serum TNC < 300 ng/ml. This result was validated in the inpatient cohort. The sensitivity and specificity of serum TNC ≥ 300 ng/ml for predicting mortality were 74.3 and 74.7% in the emergency cohort, and 63.0 and 70.1% in the inpatient cohort, respectively.Conclusion: Serum TNC was associated with mortality in critically ill patients with multiple organ dysfunction, and would be used as a prognostic tool for predicting mortality in this population.