MicroRNAs play an important role in cell proliferation and migration in hepatocellular carcinoma (HCC). In this study, the expression of MiR-331-3p in the liver cancer tissues of 116 patients, and in one normal (HL-7702) and five HCC cell lines (HepG2, SMMC-7721, Bel-7402, QGY-7701 and QGY-7703) was determined by qRT-PCR. Results indicated that MiR-331-3p was significantly repressed in HCC patients and in HCC cell lines, especially in the Bel-7402 cells when compared to normal liver tissue and the normal hepatocyte line, HL-7702. Therefore, artificial MiR-331-3p mimic computationally targeting E2F1 and anti-sense MiR-331-3p inhibitor were designed and used to transfect the Bel-7402 lines. Stable expression and significant inhibition of MiR-331-3p, with clear changes in cell morphology, proliferation and motility, were detected in Bel-7402 cells transfected with MiR-331-3p mimic or inhibitor, respectively. As E2F1 was computationally recognized as the target of MiR-331-3p, the expression of E2F1 in transfected Bel-7402 cells was analyzed by qRT-PCR and Western blotting. Results showed a strong inhibition of E2F1 by MiR-331-3p and further promotion of E2F1 by MiR-331-3p inhibitor in transfected Bel-7402 cells. Flow cytometry analysis showed that the cell ratio was restored to S to G0/1 cell number. Inhibition of E2F1 by MiR-331-3p mimic promoted cell proliferation and migration in Bel-7402. These results demonstrated that MiR-331-3p down-regulated E2F1 to promote cell proliferation and migration in HCC.