Mammary glands exhibit a series of developmental states that are typified by proliferation, differentiation, and involution. Here, we demonstrate that a microRNA (miRNA), miR-101a, plays an important role in the process of mammary gland development. We used miRNA microarray analysis to show that some miRNAs exhibit changes in their expression during mouse mammary gland epithelial cell (HC11) differentiation, which corresponds to the time when these cells acquire the milk-producing phenotype. In particular, we observed an increase of miR-101a expression throughout differentiation and involution in mammary gland tissue, as well as in HC11 cells. Overexpression experiments revealed that miR-101a suppressed the expression of beta-casein mRNA, a milk protein, and marker of cell differentiation, but its suppression was not mediated by transcriptional or direct post-transcriptional regulation of beta-casein mRNA. Overexpression of miR-101a also inhibited HC11 cell proliferation that could influence the differentiation state of the mammary gland. We speculate that a direct target of miR-101a is cyclooxygenase-2 (Cox-2) mRNA because there was an inverse relationship between these two genes during mammary gland development. Indeed, Cox-2 protein expression was suppressed by the overexpression of miR-101a, and the luciferase activity of reporter constructs containing the Cox-2 3'UTR was also suppressed by miR-101a overexpression. As Cox-2 has been shown to mediate cell proliferation, it is possible that the inhibition of HC11 cell proliferation by miR-101a might be mediated by Cox-2. Taken together, these results suggest that miR-101a regulates cell proliferation via altering Cox-2 expression, which is critical for controlling mammary gland development.