Objective: To investigate the influence of miR-101 on the biological behavior of hepatocellular carcinoma (HCC) cells. Methods: The HCC HepG2 and SMMC-7721 cells were transfected with miR-101 mimics or miRNA negative control sequences, using the two types of cells cultured without any treatment as corresponding blank control, and then the effect of miR-101 on proliferation, colony formation ability, apoptosis and cell cycle, as well as the migration and invasion ability of the two types of cells were observed. Results: Compared with corresponding blank control, in either HepG2 or SMMC-7721 cells after transfection with miR-101 minics, the proliferative ability was decreased, colony formation was reduced, G0/G1 phase cell ratio was increased, apoptosis rate was increased, and the ability of migration and invasion was reduced markedly (all P<0.05); transfection of miRNA negative control sequences exerted no obvious effect on above parameters for the two types of cells (all P>0.05). Conclusion: miR-101 possibly plays a role as cancer suppressor gene in HCC cells, and upregulating its expression may suppress the malignant activity of HCC.