Long interspersed nuclear element 1 (LINE1/L1) retrotransposons, which comprise 17% of the human genome, typically remain inactive in healthy somatic cells but are reactivated in several cancers. We previously demonstrated that p53 silences L1 transposons in human somatic cells, potentially acting as a tumor-suppressive mechanism. However, the precise molecular mechanisms underlying p53-mediated repression of L1 and its life cycle intermediates remain unclear. In this study, we used DNA-RNA immunoprecipitation-sequencing experiments to investigate RNA-DNA hybrids, which are key intermediates formed during L1 retrotransposition. Our findings reveal that L1 mRNA-genomic DNA (cis L1 R-loops) and L1 mRNA-complementary DNA (trans L1 R-loops) hybrids are upregulated in p53