Piwi-interacting RNAs (piRNAs) are a novel class of small regulatory RNAs that are expressed specifically and abundantly in germ cells. Mammalian piRNAs are 26 approximately 31 nucleotides (nt) in length and bind to Piwi proteins, but their function and biogenesis remain elusive. Here we report that the 3'-termini of mouse piRNAs are 2'-O-methylated. Furthermore, the mouse homolog of HEN1, a plant microRNA (miRNA) 2'-O-methyltransferase, is expressed specifically in testis and methylates 3'-termini of piRNAs in vitro. These findings provide insight into the biogenesis of piRNAs.