Objective: To analyze the degradome of extracellular proteins of hepatocellular carcinoma (HCC) cell line HCCLM3 through proteomic techniques, so as to screen a marker for HCC. Methods: Extracellular proteins of HCCLM3 were resolved by polyacylamide gel electrophoresis (PAGE), and were then identified by using Linear Ion Trap Quadrupole-Fourier Transform-Mass Spectrometry (LTQ-FT-MS/MS) after in-gel digestion. The degradome was analyzed through on-line database searching combined with molecular weight information. The osteopontin (OPN) expression in the plasma of healthy subjects, hepatitis B, hepatitis B-caused cirrhosis and HCC patients were quantified by Western blot method. Results: The HCCLM3 extracellular proteins were resolved into 51 bands and 1 405 non-redundant proteins were identified, Among which 474 proteins were degradated, and the secretory proteins had the highest degradation ratio (50%). The serum level of 20-kD fragment of OPN was significantly higher in HCC patients than that in other three populations. The receiver operating characteristic (ROC) showed the area under curve (AUC) of AFP for HCC diagnosis was 0.79 and 20-kD fragment of OPN was 0.68, respectively, and the combination of the two could raise the AUC to 0.89. Conclusion: The combination use of gel electrophoresis and MS is an effective and feasible approach to investigate the HCC degradome. The 20-kD fragment of OPN may be a potential marker for HCC diagnosis.