Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. Here, we report Tailor, an uridylyltransferase that is required for the majority of 3' end modifications of microRNAs in Drosophila and predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3' overhang of microRNA hairpins, which regulates processing by Dicer-1 and destabilizes RNA hairpins. Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron selectivity is explained by primary sequence specificity of Tailor, selecting substrates ending with a 3' guanosine. In contrast to mirtrons, conserved Drosophila precursor microRNAs are significantly depleted in 3' guanosine, thereby escaping regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to Tailor-directed uridylation shapes the nucleotide composition of precursor microRNA 3' ends. Hence, hairpin uridylation may serve as a barrier for the de novo creation of microRNAs in Drosophila.