Objective To study the biological behavior of human liver cancer stem cells. Methods Human liver cancer MHCC97H cells in logarithmic growth phase were inoculated in the serum free medium for suspension cultivation and human liver cancer MHCC97H stem cells with stable propagation were acquired. Human liver cancer MHCC97H stem cell microspheres and human liver cancer MHCC97H cells were collected. Messenger ribonucleic acid (mRNA) of cell markers including cluster of differentiation (CD) 133, CD90, epithelial cell adhesion molecule (EpCAM) was detected by reverse transcription polymerase chain reaction (RT-PCR). Colony formation assay, tumor cell invasion assay (Transwell assay) and nude mouse tumorigenicity assay were performed respectively to observe the colony formation, cell invasion and tumorigenicity of two kinds of cells. The experimental data of two kinds of cells were compared by t test or t′test. Results The mean contents of CD133, CD90, EpCAM mRNA in human liver cancer MHCC97H stem cells (32.70±0.20, 66.30±0.32, 115.40±4.00) were significantly higher than those in human liver cancer MHCC97H cells (1.27±0.29, 13.60±1.36, 1.34±0.40) (t′=117.8, 53.97, 83.37;P<0.05). The colony forming efifciency was (213±10)%in human liver cancer MHCC97H stem cells and was (54±11)%in human liver cancer MHCC97H cells, where signiifcant difference was observed (t=13.11, P<0.05). Through the Transwell assay, the membrane permeating cell count was (587±120)/visual ifelds in human liver cancer MHCC97H stem cells, and was (97±13)/visual ifelds in human liver cancer MHCC97H cells, where signiifcant difference was observed (t=6.38, P<0.05). In the nude mouse tumorigenicity assay, subcutaneous transplanted tumors were observed in nude mouse 4 weeks after inoculating 2×103 human liver cancer MHCC97H stem cells and the tumor formation rate was 1/6. Subcutaneous transplanted tumors were observed in all nude mice by inoculating 2×105 human liver cancer MHCC97H stem cells, and the tumor formation rate was 6/6. Subcutaneous transplanted tumors were observed in nude mice by inoculating at least 2×105 human liver cancer MHCC97H cells, and the tumor formation rate was 2/6. Conclusion Compared with the human liver cancer MHCC97H cells, human liver cancer MHCC97H stem cells possess stronger invasion and higher tumor formation rate.