miR-122 is a highly abundant, hepatocyte-specific microRNA. The biomedical significance and regulatory mechanisms of miR-122 remain obscure. We explored the role of miR-122 in tumorigenesis in the context of gene regulatory network. The miR-122 promoter and its transactivator were identified by way of luciferase reporter system, electrophoretic mobility shift, and chromatin immunoprecipitation assays. The miR-122 regulatory circuitry and its implication in hepatocarcinogenesis were identified using livers of different development stages, human hepatocellular carcinoma (HCC) tissues and cell lines, and aflatoxin B1 (AFB1)-transformed cells. We characterized the −5.3 to −4.8 kb region upstream of miR-122 precursor as miR-122 promoter. Further investigation revealed that deletion of predicted CCAAT/enhancer-binding protein alpha (C/EBPα) binding sites C/EBPα knockdown significantly reduced miR-122 promoter activity and endogenous miR-122 expression; and C/EBPα directly interacted with the miR-122 promoter in vitro and in vivo . These data suggest that C/EBPα is a transactivator for miR-122 transcription. We further demonstrated that miR-122 suppressed insulin-like growth factor 1 receptor (IGF-1R) translation and sustained glycogen synthase kinase-3 beta (GSK-3β) activity. The activated GSK-3β not only repressed cell proliferation, but also activated C/EBPα, which maintained miR-122 levels and thereby enforced IGF-1R suppression. Interestingly, down-regulation of miR-122 and C/EBPα, and up-regulation of IGF-1R were frequently observed in HCC tissues, and decreased miR-122 levels were associated with worse survival of HCC patients. Moreover, AFB1 exposure resulted in decreased activity in GSK-3β, C/EBPα, and miR-122 and increased levels of IGF-1R, whereas restoration of miR-122 suppressed the tumorigenicity of HCC and AFB1-transformed cells. Conclusion: We have identified a novel GSK-3β–C/EBPα–miR-122–IGF-1R regulatory circuitry whose dysfunction may contribute to the development of HCC. Our findings provide new insight into miR-122's function and the mechanisms of hepatocarcinogenesis. (Hepatology 2010;52:1702-1712)