Wylie et al. show that p53 limits retrotransposition and interacts with members of the piwi-interacting RNA (piRNA) pathway in Drosophila melanogaster and zebrafish. Normal human TP53 alleles suppressed retrotransposition but cancer-associated TP53 mutants did not. Unrestrained retrotransposition was also observed in mouse and human cancers with p53 alterations, indicating that this ancestral function of p53 to suppress retrotransposition may also suppress tumorigenesis.