MicroRNAs are tiny RNA molecules which serve as important post-transcriptional regulators of gene expression. Dysregulated expression of microRNAs has been observed in human cancers, indicating that microRNAs may function as oncogenes or as tumor suppressors. To date, the microRNAs encoded by the oncogenic miR-17-92 cluster, and its paralog the miR-106b-25 cluster, are among those which are differentially expressed in human cancers. In this study, we examined and confirmed the over-expression of these clusters in hepatocellular carcinoma and in hepatoma-derived cells. At least 50% of the tumor samples showed a greater than two-fold increase in the expression for miR-18 and for the miR-106b-25 cluster when compared with the corresponding paired non-tumor samples. Knock-down studies for the miR-106b-25 cluster, which includes miR-106b, miR-93 and miR-25, showed that the expression of the cluster is necessary for cell proliferation and for anchorage-independent growth. In tumors with high expression of this cluster, reduced expression of the BH3-only protein Bim, a miR-25 target, was observed. We further identified the transcription factor E2F1 as a target gene for miR-106b and miR-93 and it is likely that one of the roles of the miR-106b-25 cluster is to prevent excessively high E2F1 expression, which may then cause apoptosis. We conclude that there is aberrant expression of microRNAs encoded by the oncogenic miR-17-92 cluster and the miR-106b-25 cluster in hepatocellular carcinoma. The consistent overexpression of the miR-106b-25 cluster and its role in cell proliferation and anchorage-independent growth points to the oncogenic potential of this cluster. (Cancer Sci 2009; 100: 1234–1242)