Argonaute proteins are categorized into AGO and PIWI clades. Across most animal species, AGO-clade proteins are widely expressed in various cell types, and regulate normal gene expression1. By contrast, PIWI-clade proteins predominantly function during gametogenesis to suppress transposons and ensure fertility1,2. Both clades use nucleic acid guides for target recognition by means of base pairing, crucial for initiating target silencing, often through direct cleavage. AGO-clade proteins use a narrow channel to secure a tight guide–target interaction3. By contrast, PIWI proteins feature a wider channel that potentially allows mismatches during pairing, broadening target silencing capability4,5. However, the mechanism of PIWI-mediated target cleavage remains unclear. Here we demonstrate that after target binding, PIWI proteins undergo a conformational change from an ‘open’ state to a ‘locked’ state, facilitating base pairing and enhancing target cleavage efficiency. This transi