Genotoxic stress during DNA replication constitutes a serious threat to genome integrity and causes human diseases. Defects at different steps of DNA metabolism are known to induce replication stress, but the contribution of other aspects of cellular metabolism is less understood. We show that aminopeptidase P (APP1), a metalloprotease involved in the catabolism of peptides containing proline residues near their N-terminus, prevents replication-associated genome instability. Functional analysis of C. elegans mutants lacking APP-1 demonstrates that germ cells display replication defects including reduced proliferation, cell cycle arrest, and accumulation of mitotic DSBs. Despite these defects, app-1 mutants are competent in repairing DSBs induced by gamma irradiation, as well as SPO-11-dependent DSBs that initiate meiotic recombination. Moreover, in the absence of SPO-11, spontaneous DSBs arising in app-1 mutants are repaired as inter-homologue crossover events during meiosis, confirming that APP-1 is not required for homologous recombination. Thus, APP-1 prevents replication stress without having an apparent role in DSB repair. Depletion of APP1 (XPNPEP1) also causes DSB accumulation in mitotically-proliferating human cells, suggesting that APP1's role in genome stability is evolutionarily conserved. Our findings uncover an unexpected role for APP1 in genome stability, suggesting functional connections between aminopeptidase-mediated protein catabolism and DNA replication. Author summaryThe accurate duplication of DNA that occurs before cells divide is an essential aspect of the cell cycle that is also crucial for the correct development of multicellular organisms. Mutations that compromise the normal function of the DNA replication machinery can lead to the accumulation of replication-related DNA damage, a known cause of human disease and a common feature of cancer and precancerous cells. Therefore, identifying factors that prevent replication-related DNA damage is highly relevant for human health. In this manuscript, we identify aminopeptidase P, an enzyme involved in the breakdown of proteins containing the amino acid Proline at their N-terminus, as a novel factor that prevents replication-related DNA damage. Analysis of C. elegans nematodes lacking aminopeptidase P reveals that this protein is required for normal fertility and development, and that in its absence proliferating germ cells display DNA replication defects, including cell cycle arrest and accumulation of extensive DNA damage. We also show that removal of aminopeptidase P induces DNA damage in proliferating human cells, suggesting that its role in preventing replication defects is evolutionarily conserved. These findings uncover functional connections between aminopeptidase-mediated protein degradation and DNA replication.