The epithelium of the intestinal mucosa is a rapidly self-renewing tissue in the body, and defects in the renewal process occur commonly in various disorders. microRNAs (miRNAs) posttranscriptionally regulate gene expression and are implicated in many aspects of cellular physiology. Here we investigate the role of miRNA-29b (miR-29b) in the regulation of normal intestinal mucosal growth and further validate its target mRNAs. miRNA expression profiling studies reveal that growth inhibition of the small intestinal mucosa is associated with increased expression of numerous miRNAs, including miR-29b. The simple systemic delivery of locked nucleic acid–modified, anti–miR-29b-reduced endogenous miR-29b levels in the small intestinal mucosa increases cyclin-dependent kinase 2 (CDK2) expression and stimulates mucosal growth. In contrast, overexpression of the miR-29b precursor in intestinal epithelial cells represses CDK2 expression and results in growth arrest in G1 phase. miR-29b represses CDK2 translation through direct interaction with the cdk2 mRNA via its 3′-untranslated region (3′-UTR), whereas point mutation of miR-29b binding site in the cdk2 3′-UTR prevents miR-29b–induced repression of CDK2 translation. These results indicate that miR-29b inhibits intestinal mucosal growth by repressing CDK2 translation.