Hepatocellular carcinoma (HCC) remains a significant unmet medical need with very limited therapeutic options available. Although microRNA-21 (miR-21) has been shown to be upregulated in HCC, its contribution as an onco-miR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single- stranded oligonucleotide inhibitors of miR-21 (anti-miR-21) and used them to interrogate dependency on miR-21 in a panel of 20 commercially available HCC cell lines in vitro. Upon lipid-mediated transfection, anti-miR-21, but not its mismatched (MM) control, caused significant de-repression of known direct targets of miR-21, inhibited survival of a large subset of HCC cell lines. Sensitive HCC cell lines showed dose- and time-dependent induction of caspase 3/7 activity upon treatment with anti-miR-21. In contrast, non- responder HCC cell lines failed to significantly upregulate caspase activity and maintained viability in the presence of anti-miR compound. To better understand the consequences of miR-21 suppression in HCC, we carried out global gene expression profiling of anti-miR-21 treated sensitive liver cancer cells. Striking enrichment in miR-21 targets was noted among upregulated transcripts. Key cellular processes affected by miR-21 inhibition, including deregulation of metabolic pathways, were identified by gene ontology analysis. In summary, our data suggest that inhibition of miR-21 merits further investigation in the treatment of hepatocellular carcinoma.