LIN28 (a homologue of the Caenorhabditis elegans lin-28 gene) is an evolutionarily conserved RNA-binding protein and a master regulator controlling the pluripotency of embryonic stem cells. Together with OCT4, SOX2, and NANOG, LIN28 can reprogram somatic cells, producing induced pluripotent stem cells. Expression of LIN28 is highly restricted to embryonic stem cells and developing tissues. In human tumors, LIN28 is up-regulated and functions as an oncogene promoting malignant transformation and tumor progression. However, the mechanisms of transcriptional and post-transcriptional regulation of LIN28 are still largely unknown. To examine microRNAs (miRNAs) that repress LIN28 expression, a combined in silico prediction and miRNA library screening approach was used in the present study. Four miRNAs directly regulating LIN28 (let-7, mir-125, mir-9, and mir-30) were initially identified by this approach and further validated by quantitative RT-PCR, Western blot analysis, and a LIN28 3'-UTR reporter assay. We found that expression levels of these four miRNAs were clustered together and inversely correlated with LIN28 expression during embryonic stem cell differentiation. In addition, the expression of these miRNAs was remarkably lower in LIN28-positive tumor cells compared with LIN28-negative tumor cells. Importantly, we demonstrated that these miRNAs were able to regulate the expression and activity of let-7, mediated by LIN28. Taken together, our studies demonstrate that miRNAs let-7, mir-125, mir-9, and mir-30 directly repress LIN28 expression in embryonic stem and cancer cells. Global down-regulation of these miRNAs may be one of the mechanisms of LIN28 reactivation in human cancers.