Distinguishing self from non-self plays a crucial role in safeguarding the germlines of metazoa from mobile DNA elements. Since their discovery less than a decade ago, Piwi-interacting RNAs (piRNAs) have been shown to repress transposable elements in the germline and, hence, have been at the forefront of research aimed at understanding the mechanisms that maintain germline integrity. More recently, roles for piRNAs in gene regulation have emerged. In this Review, we highlight recent advances made in understanding piRNA function, highlighting the divergent nature of piRNA biogenesis in different organisms, and discussing the mechanisms of piRNA action during transcriptional regulation and in transgenerational epigenetic inheritance.