Variations in microRNA (miRNA) gene and/or target repertoire are likely to be key drivers of phenotypic differences between species. To better understand these changes, we developed a computational method that identifies signatures of species-specific target site gain and loss associated with miRNA acquisition. Interestingly, several of the miRNAs implicated in mouse 3' UTR evolution derive from a single rapidly expanded rodent-specific miRNA cluster. Located in the intron of Sfmbt2, a maternally imprinted polycomb gene, these miRNAs (referred to as the Sfmbt2 cluster) are expressed in both embryonic stem cells and the placenta. One abundant miRNA from the cluster, miR-467a, functionally overlaps with the mir-290-295 cluster in promoting growth and survival of mouse embryonic stem cells. Predicted novel targets of the remaining cluster members are enriched in pathways regulating cell survival. Two relevant species-specific target candidates, Lats2 and Dedd2, were validated in cultured cells. We suggest that the rapid evolution of the Sfmbt2 cluster may be a result of intersex conflict for growth regulation in early mammalian development and could provide a general model for the genomic response to acquisition of miRNAs and similar regulatory factors.