PIWI-interacting RNAs (piRNAs) silence transposons to maintain genome integrity in animal germ lines(1-4). piRNAs are classified as primary and secondary piRNAs, depending on their biogenesis machinery(5-10). Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters in the genome through the primary processing pathway(5,8-10). Although the existence of a ribonuclease participating in this pathway has been predicted, its molecular identity remained unknown. Here we show that Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily member(11), is an endoribonuclease essential for primary piRNA biogenesis. We solved the crystal structure of Drosophila melanogaster Zuc (DmZuc) at 1.75 angstrom resolution. The structure revealed that DmZuc has a positively charged, narrow catalytic groove at the dimer interface, which could accommodate a single-stranded, but not a double-stranded, RNA. DmZuc and the mouse homologue MmZuc (also known as Pld6 and MitoPLD)(12-14) showed endoribonuclease activity for single-stranded RNAs in vitro. The RNA cleavage products bear a 5'-monophosphate group, a hallmark of mature piRNAs. Mutational analyses revealed that the conserved active-site residues of DmZuc are critical for the ribonuclease activity in vitro, and for piRNA maturation and transposon silencing in vivo. We propose a model for piRNA biogenesis in animal germ lines, in which the Zuc endoribonuclease has a key role in primary piRNA maturation.