Osteopontin (OPN) is a secreted phosphorylated and glycosylated protein, which plays an important role in carcinogenesis and metastasis. In hepatocellular carcinoma (HCC), OPN is being investigated either as a therapeutic target gene or as a biomarker for diagnosis. Yet, the role of the anti-OPN autoantibody in HCC remains unclear. In the present study, the level of serum anti-OPN autoantibody in HCC was analyzed by enzyme-linked immunosorbent assay. Immunohistochemistry (IHC) was also performed to analyze protein expression profiles and the prognostic significance of OPN in HCC. In this study, the prevalence and titer of anti-OPN autoantibodies in HCC were significantly higher than these values in normal human serum (NHS) (P=0.001, P=0.000, respectively). When both α-fetoprotein and the autoantibody against OPN were used simultaneously as diagnostic biomarkers, the sensitivity was up to 65%. In IHC, 59 of the 83 (65.6%) HCC specimens expressed OPN with cytoplasmic positive staining. The overall survival (OS) of HCC patients with OPN-positive tumors was 28.81 months compared to 39.37 months for HCC patients with OPN-negative tumors (P<0.01). Furthermore, multivariate analysis showed that OPN overexpression was the strongest independent adverse prognostic factor for OS (P=0.02). Taken together, our data indicate that the anti-OPN autoantibody may be a supplementary serological biomarker for HCC, and is correlated with poor prognosis in HCC patients.