Increasing evidence indicates that microRNAs (miRNAs) play important roles in mouse brain development. We and several other reports recently have demonstrated that Wnt1-cre-mediated loss of Dicer, the key enzyme for miRNA biosynthesis, results in malformation of the midbrain and cerebellum and failure of neural crest and dopaminergic differentiation. The underlying mechanisms, however, remain poorly understood. The resemblance of some of the phenotypes in the Wnt1-cre Dicer conditional knockout embryos and Wnt1(-/-),Wnt1(-/-);Wnt3(-/-) and Wnt1-cre;beta-cateninn(flox/flox) knockout embryos reminds us that loss of miRNA may disrupt the Wnt-beta-catenin signaling. Here we provide evidence that miRNAs modulate the Wnt signaling pathway through targeting its inhibitors. First, we predicted miRNA binding sites in the 3' UTRs of candidate inhibitors of the Wnt signaling pathway and luciferase assays revealed that several inhibitors of Wnt signaling pathway were targeted by miRNAs. Second, we demonstrated that several miRNAs could modulate the expression of Gsk3b. an inhibitor of Wnt signaling, post-transcriptional in 293T cells. Third, we found that several miRNAs were able to regulate the Wnt-beta-catenin signaling activity in 293T cells. More interestingly, the expression of beta-catenin protein was dramatically reduced in the Wnt1-cre-meidiated Dicer knockout brain tissue compared with control. Our studies therefore suggest that miRNAs might exert their functions, at least in part, by modulating the Wnt signaling pathway through targeting its inhibitors. (C) 2011 Elsevier Inc. All rights reserved.