Vertebrate genomes each encode hundreds of micro-RNAs (miRNAs), yet for few of these miRNAs is there empirical evidence as to which mRNA(s) they regulate. Here we report the identification of human lin-28 mRNA as a regulatory target of human miR-125b and its homolog miR-125a. Studies of miR-125b function in mouse P19 embryonal carcinoma cells induced to develop into neurons suggest a role for this regulatory miRNA in mammalian neuronal differentiation, since its increased concentration in these cells contributes to lin-28 downregulation. Within the lin-28 3' untranslated region (UTR) are two conserved miRNA responsive elements (miREs) that mediate repression by miR-125b and miR-125a. Simultaneous deletion of both miREs renders the lin-28 3' UTR almost completely insensitive to these miRNAs, indicating that these two miREs are the principal elements in the lin-28 3' UTR that respond to miR-125. At the 3' end of each element is an adenosine residue that makes a significant contribution to function irrespective of its complementarity to the 5'-terminal nucleotide of miR-125. By contrast to most earlier reports of gene repression by other miRNAs that are imperfectly complementary to their targets, lin-28 downregulation by miR-125 involves reductions in both translational efficiency and mRNA abundance. The decrease in the mRNA concentration is achieved by a posttranscriptional mechanism that is independent of the inhibitory effect on translation.