Portal vein invasion (PVI) is a hallmark of metastatic potential of hepatocellular carcinoma (HCC) and is frequently found at a stage of moderately differentiated HCC. To identify genes involved in PVI of HCC associated with hepatitis C virus (HCV), we performed a comprehensive analysis of 12,600 genes in 35 moderately differentiated HCV-related HCCs by DNA microarray. Our supervised learning method identified 35 genes involved in PVI. Among the 35 identified genes, we focused on the inhibitor of DNA binding 2 (ID2), because it encodes a liver-rich dominant-negative helix-loop-helix protein. The microarray results for ID2 were reproduced by quantitative real-time reverse transcription (QRT)-PCR and Western blot analyses. In an independent set of HCV-related HCCs (n=28) and HCV-unrelated HCCs (n=14), our QRT-PCR showed that decrease in ID2 mRNA levels were associated with PVI in HCV-related HCC but not HCV-unrelated HCC. In conclusion, our results strongly suggest that ID2 plays an important role in PVI process of HCV-related HCC.