Portal vein tumor thrombosis (PVTT) in patients with hepatocellular carcinoma (HCC) is known as a major complication associated with poor survival. We clinically defined a type of distinct PVTT (dPVTT) in small HCC patients that is distant to liver parenchyma tumor (PT). The biological features of dPVTT are not clear. We utilized two-dimensional electrophoresis and tandem MS to compare and identify differentially expressed proteins between dPVTT and PT tissues. Of the 65 spots identified as differentially expressed (p < 0.05) between the two cancerous tissues, 19 (corresponding to 19 unique proteins) were identified. Further analysis of five proteins confirmed quantitative differences between the two tumor tissues. Upon comparison with PT tissues of HCC, c-kit was also significantly upregulated in dPVTTs in small HCC patients and the CSQT-2 cell line derived from dPVTT tissues, which validated the differences between the dPVTT and PT tissues. The protein expression profiles and proteins identified in this study demonstrate the presence of dPVTTs with more malignant phenotypes and will be useful in clarifying the mechanisms through which dPVTT develops. Specific treatments targeting dPVTT might be applied to HCC patients with dPVTT.