We have analysed the role of a microRNA, miR164, in boundary formation during organ initiation from Arabidopsis meristems. The establishment and maintenance of the boundary domain are controlled by three partially redundant genes, CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3. We show that miR164 overexpression phenocopies the cuc1 cuc2 double mutant by inducing post-transcriptional downregulation of CUC1 and CUC2 but not CUC3 mRNA levels. Disruption of CUC2 regulation by miR164, either by making CUC2 resistant to the miRNA or by reducing miRNA levels leads to similar enlarged boundary domains. We relate this enlargement to the division patterns of the boundary cells. We propose that miR164 constrains the expansion of the boundary domain, by degrading CUC1 and CUC2 mRNAs.