Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is associated with various diseases. We evaluated the diagnostic and prognostic significance of serum ITIH4 levels in healthy controls and patients with chronic hepatitis B (CHB), hepatitis B virus (HBV)-related liver cirrhosis, and HBV-related hepatocellular carcinoma (HCC). The study enrolled 300 individuals (50 healthy controls, 50 with CHB, 100 with HBV-associated cirrhosis, and 100 with HBV-associated HCC). Serum ITIH4 levels were determined by western blot analysis and expressed in densitometry units (DU). ITIH4 levels were higher in CHB (mean: 252.96 DU) and liver cirrhosis (mean: 206.43 DU) patients than in healthy controls (mean: 75.92 DU) and HCC patients (mean: 92.86 DU) (P<0.001). The area under the receiver operating characteristic curve was 0.71 for the diagnosis of HCC in patients with HBV-related liver disease. Multivariate Cox regression analysis showed that large tumor size (≥5cm) was independently associated with overall survival (hazard ratio 5.894, 95% confidence interval 1.373–25.300, P=0.017). A Kaplan–Meier survival analysis showed significantly worse survival among HCC patients with both low ITIH4 (<80 DU) and a large tumor size compared to that among other HCC patients (P<0.001), and among patients with high AFP (>200ng/mL) and low ITIH4 compared to that among other HCC patients (P=0.041). Serum ITIH4 levels are reduced in HCC patients compared to that in CHB and cirrhosis patients, and low serum ITIH4 levels are associated with shorter survival in HBV-associated HCC patients. •Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is associated with HCC development.•Serum ITIH4 levels are reduced in HCC compared to that in CHB and cirrhosis.•Low serum ITIH4 levels are associated with shorter survival in HCC patients.