Objective To identify, isolate, and evaluate cells with side population phenotype (SP, with the property of stem cells) from hepatocellular carcinoma. Methods Capable of pumping fluorochrome Hoechst33342 outside of cells, SP cells were isolated and purified by flow cytometry. Results We found that the ratios of SP cells in PLC,Huh7,Hep3B,SMMC7721 cell line and an HCC specimens were 0.8%, 0.3%,0.6%,0.3%,and 0.4%, respectively. We did not find SP cells in HepG2. NOD-SCID subcutaneous transplant experiments showed that SP cells had a high ability of tumorigenicity than non-SP cells. Conclusion Hepatocellular carcinoma cell lines and specimens contain a small number of SP cells, which have a higher tumorigenicity than non-SP cells; this finding paves a way for further analysis of the stem-like property of SP cells.