microRNAs play a critically important role in a wide array of biological processes including those implicated in cancer, neuro-degenerative and metabolic disorders, and viral infection. Although we have begun to understand microRNA biogenesis and function, experimental demonstration of their functional effects and the molecular mechanisms by which they function remains a challenge. Members of the let-7/miR-98 family play a critical role in cell cycle control with respect to differentiation and tumorigenesis. In this study, we show that exogenous addition of pre-let-7 in primary human fibroblasts results in a decrease in cell number and an increased fraction of cells in the G(2)/M cell cycle phase. Combining microarray techniques with DNA sequence analysis to identify potential let-7 targets, we discovered 838 genes with a let-7 binding site in their 3'-untranslated region that were down-regulated upon overexpression of let-7b. Among these genes is cdc34, the ubiquitin-conjugating enzyme of the Skp1/cullin/F-box (SCF) complex. Cdc34 protein levels are strongly down-regulated by let-7 overexpression. Reporter assays demonstrated direct regulation of the cdc34 3'-untranslated region by let-7. We hypothesized that low Cdc34 levels would result in decreased SCF activity, stabilization of the SCF target Wee1, and G(2)/M accumulation. Consistent with this hypothesis, small interfering RNA-mediated down-regulation of Wee1 reversed the G(2)/M phenotype induced by let-7 overexpression. We conclude that Cdc34 is a functional target of let-7 and that let-7 induces down-regulation of Cdc34, stabilization of the Wee1 kinase, and an increased fraction of cells in G(2)/M in primary fibroblasts.