AIM:To determine the prevalence and diagnostic value of autoantibodies in α-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC). METHODS:Fifty-six serum samples from AFP-negative HCC cases, 86 from AFP-positive HCC cases, 168 from chronic liver disease cases, and 59 from normal human controls were included in this study. Autoantibodies to nucleophosmin (NPM)1, 14-3-3zeta and mouse double minute 2 homolog (MDM2) proteins in AFP-negative HCC serum were evaluated by enzyme-linked immunosorbent assay. Partially positive sera were further evaluated by western blotting. Immunohistochemistry was used to detect the expression of three tumor-associated antigens (TAAs) in AFP-negative HCC and normal control tissues. RESULTS:The frequency of autoantibodies to the three TAAs in AFP-negative HCC sera was 21.4%, 19.6% and 19.6%, which was significantly higher than in the chronic liver disease cases and normal human controls ( < 0.01) as well as AFP-positive HCC cases. The sensitivity of the three autoantibodies for diagnosis of AFP-negative HCC ranged from 19.6% to 21.4%, and the specificity was approximately 95%. When the three autoantibodies were combined, the sensitivity reached 30.4% and the specificity reached 91.6%. CONCLUSION:Autoantibodies to NPM1, 14-3-3zeta and MDM2 may be useful biomarkers for immunodiagnosis of AFP-negative HCC.