Piwi-interacting RNAs (piRNAs) are a germline-specific class of small noncoding RNAs that are essential for spermatogenesis, but their function and biogenesis remain elusive. Here we report a post-transcriptional modification of mouse piRNAs. Mass spectrometric analysis reveals that the piRNAs tested are fully modified by 2'-O-methylation at their 3' termini. This observation may provide a clue to the biogenesis and function of piRNAs in spermatogenesis.