Peripheral myelin protein 22 (PMP22) is a dose-sensitive, disease-associated protein primarily expressed in myelinating Schwann cells. Either reduction or overproduction of PMP22 can result in hereditary neuropathy, suggesting a requirement for correct protein expression for peripheral nerve biology. PMP22 is post-transcriptionally regulated and the 3′untranslated region (3′UTR) of the gene exerts a negative effect on translation. MicroRNAs (miRNAs) are small regulatory molecules that function at a post-transcriptional level by targeting the 3′UTR in a reverse complementary manner. We used cultured Schwann cells to demonstrate that alterations in the miRNA biogenesis pathway affect PMP22 levels, and endogenous PMP22 is subjected to miRNA regulation. GW-body formation, the proposed cytoplasmic site for miRNA-mediated repression, and Dicer expression, an RNase III family ribonuclease involved in miRNA biogenesis, are co-regulated with the differentiation state of Schwann cells. Furthermore, the levels of Dicer inversely correlate with PMP22, while the inhibition of Dicer leads to elevated PMP22. Microarray analysis of actively proliferating and differentiated Schwann cells, in conjunction with bioinformatics programs, identified several candidate PMP22-targeting miRNAs. Here we demonstrate that miR-29a binds and inhibits PMP22 reporter expression through a specific miRNA seed binding region. Over-expression of miR-29a enhances the association of PMP22 RNA with Argonaute 2, a protein involved in miRNA function, and reduces the steady-state levels of PMP22. In contrast, inhibition of endogenous miR-29a relieves the miRNA-mediated repression of PMP22. Correlation analyses of miR-29 and PMP22 in sciatic nerves reveal an inverse relationship, both developmentally and in post-crush injury. These results identify PMP22 as a target of miRNAs and suggest that myelin gene expression by Schwann cells is regulated by miRNAs. © 2009 Wiley-Liss, Inc.