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Human beta-defensin-1 (hBD-1) is a natural antimicrobial peptide expressed in the epithelia of multiple tissues including the digestive tract. In the current study, hBD-1 levels were determined in different subsets of patients with decompensated cirrhosis including acute-on-chronic liver failure (ACLF). In addition, the association with mortality of hBD-1, C-reactive protein (CRP) and procalcitonin (PCT) was assessed.A total of 125 patients were divided into three groups: 39 with ACLF (derivation cohort), 46 with acute decompensation without ACLF (AD) and 40 with decompensated cirrhosis without an acute event (DC). The data from 24 different ACLF patients were used for validation and 15 healthy individuals as control group.Serum hBD-1, CRP and PCT levels were higher in ACLF compared to both AD and DC groups (P < 0.001). Healthy controls demonstrated similar hBD-1 and PCT values compared to DC group. In ROC curve, the performance of hBD-1 to predict 60-day mortality in ACLF group was similar in derivation and validation cohorts (c-statistic 0.834 and 0.879, respectively). CRP was a poor predictor of mortality. In ACLF group, patients with high hBD-1 (>36.625 ng/mL) had a poor prognosis at 60 days compared to those with lower values (log-rank P = 0.001). In Cox multivariate regression analysis, only hBD-1 (HR 1.020, 95%CI 1.006–1.035, P = 0.006) emerged as an independent predictor of death in ACLF group. In AD group, neither hBD-1 nor PCT or CRP variables were associated with mortality.High hBD-1 was detected at presentation in patients with ACLF who died during follow-up period. hBD-1 is an accurate predictor of short-term mortality in patients with ACLF.