PIWI-interacting RNAs (piRNAs) are a class of small silencing RNAs that play a crucial role in preserving the germline genomic integrity by silencing transposable elements (TEs). As the most heterogeneous small RNAs, they comprise over one million unique sequences, enabling them to comprehensively target and repress the diverse TE families in each species and thus safeguard the genome. piRNA biogenesis is distinct from other small RNA pathways, relying on a unique set of proteins and processing from long single-stranded RNA precursors. While many core components of the piRNA pathway are evolutionarily conserved, different animal clades, including Tetrahymena, nematodes, arthropods, and amniotes, have evolved clade-specific strategies for piRNA production. Functionally, beyond their well-established role in TE silencing at both transcriptional and post-transcriptional levels, piRNA pathways are increasingly recognized for their broader, species-specific regulatory functions. These include controlling gene expression in germ cells and supporting diverse somatic roles, particularly in invertebrates. This chapter provides a comparative overview of piRNA biogenesis and function in Drosophila and mice, highlighting species-specific adaptations and detailing their molecular mechanisms in defending against TE activation. A deeper understanding of piRNA pathways not only sheds light on their fundamental role in the evolutionary arms race with TEs, a conflict that dates back to the origin of life, but also reveals their emerging relevance in human infertility.