microRNAs (miRNAs) are small RNAs that in general down-regulate the intracellular abundance and translation of targetmRNAs. We noted that sequestration of liver-specific miR-122 by modified antisense oligonucleotides resulted in a dramaticloss of hepatitis C virus (HCV) RNA in cultured human liver cells. A binding site for miR-122 was predicted to reside closeto the 5′ end of the viral genome, and its functionality was tested by mutational analyses of the miRNA-binding site in viralRNA, resulting in reduced intracellular viral RNA abundance. Importantly, ectopic expression of miR-122 molecules that containedcompensatory mutations restored viral RNA abundance, revealing a genetic interaction between miR-122 and the viralRNA genome. Studies with replication-defective viral RNAs demonstrated that miR-122 affected mRNA abundance by positivelymodulating RNA replication. In contrast, interaction of miR-122 with the 3′-noncoding region (3′ NCR) of the cellularmRNA encoding the cationic amino acid transporter CAT-1 resulted in the down-regulation of CAT-1 protein abundance. These findings provide evidence that a specific miRNA can regulate distinct target mRNAs in both a positive and negativefashion. The positive role of miR-122 in viral replication suggests that this miRNA could be targeted for antiviral therapy.