Significance In this study, we identified the direct interaction region between Drosophila Piwi and Papi. We further determined the crystal structures of Papi-eTud in the apo form, in complex with unmethylated Piwi peptide, and in complex with symmetrically dimethylated Piwi peptide at arginine-10, which demonstrated that Papi interacts with an RGRRR motif on the N terminus of Piwi in a sequence-specific manner both in vitro and in vivo. This recognition sequence, which determines the specificity of Papi–Piwi interactions, is different from all previously reported (G/A)R repeats. Our studies provide mechanistic insights into the important role of Papi–Piwi interactions in the 3′ end-trimming process of PIWI-interacting RNA biogenesis and facilitate the identification of new PIWI-interacting partners of Tudor domain-containing proteins.