During microRNA (miRNA) biogenesis, one strand of a approximately 21-22-nucleotide RNA duplex is preferentially selected for entry into a silencing complex. The other strand, known as the miRNA* species, has typically been assumed to be a carrier strand. Here we show that, although Drosophila melanogaster miRNA* species are less abundant than their partners, they are often present at physiologically relevant levels and can associate with Argonaute proteins. Comparative genomic analyses revealed that >40% of miRNA* sequences resist nucleotide divergence across Drosophilid evolution, and at least half of these well-conserved miRNA* species select for conserved 3' untranslated region seed matches well above background noise. Finally, we validated the inhibitory activity of miRNA* species in both cultured cells and transgenic animals. These data broaden the reach of the miRNA regulatory network and suggest an important mechanism that diversifies miRNA function during evolution.