MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression with functional links to tumorigenesis. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, and it is heterogeneous in clinical outcomes and biological activities. Recently, we have identified a subset of highly invasive epithelial cell adhesion molecule (EpCAM)⁺ HCCcells from alpha-fetoprotein (AFP)⁺ tumors with cancer stem/progenitor cell features, that is, the abilities to self-renew, differentiate, and initiate aggressive tumors in vivo. Here, using a global microarray-based miRNA profiling approach followed by validation with quantitative reverse transcription polymerase chain reaction, we have demonstrated that conserved miR-181 family members were up-regulated in EpCAM⁺AFP⁺ HCCs and in EpCAM⁺ HCC cells isolated from AFP⁺ tumors. Moreover, miR-181 family members were highly expressed in embryonic livers and in isolated hepatic stem cells. Importantly, inhibition of miR-181 led to a reduction in EpCAM⁺ HCC cell quantity and tumor initiating ability, whereas exogenous miR-181 expression in HCC cells resulted in an enrichment of EpCAM⁺ HCC cells. We have found that miR-181 could directly target hepatic transcriptional regulators of differentiation (for example, caudal type homeobox transcription factor 2 [CDX2] and GATA binding protein 6 [GATA6]) and an inhibitor of Wnt/β-catenin signaling (nemo-like kinase [NLK]). Taken together, our results define a novel regulatory link between miR-181s and human EpCAM⁺ liver cancer stem/progenitor cells and imply that molecular targeting of miR-181 may eradicate HCC.