PIWI-interacting RNAs (piRNAs) are small RNAs required to recognize and silence transposable elements. The 5' ends of mature piRNAs are defined through cleavage of long precursor transcripts, primarily by Zucchini (Zuc). Zuc-dependent cleavage typically occurs immediately upstream of a uridine. However, Zuc lacks sequence preference in vitro, pointing towards additional unknown specificity factors. Here, we examine murine piRNAs and reveal a strong and specific enrichment of three sequences (UAA, UAG, UGA)-corresponding to stop codons-at piRNA 5' ends. Stop codon sequences are also enriched immediately after piRNA processing intermediates, reflecting their Zuc-dependent tail-to-head arrangement. Further analyses reveal that a Zuc in vivo cleavage preference at four sequences (UAA, UAG, UGA, UAC) promotes 5' end stop codons. This observation is conserved across mammals and possibly further. Our work provides new insights into Zuc-dependent cleavage and may point to a previously unrecognized connection between piRNA biogenesis and the translational machinery.