ABSTRACT Introduction Inhibitors of poly(ADP-ribose) polymerase (PARP) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, resistance to PARP inhibitors (PARPi) is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. Material and methods To tackle this question, we generated matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumours. By combining next-generation sequencing with functional genetic screens, we identified loss of poly(ADP-ribose) glycohydrolase (PARG) as a major resistance mechanism. Results and discussions We demonstrate that PARG depletion restores PAR formation, rescues controlled DNA replication fork progression and promotes the recruitment of downstream DNA repair factors. The potential relevance of PARG in clinical PARPi resistance is underscored by the presence of PARG-negative clones in a subset of human triple-negative breast and serous ovarian cancers. Importantly, acquisition However, the gain of PARPi resistance comes at a cost, as PARG inactivation results in new vulnerabilities that can be exploited therapeutically. Conclusion We conclude that loss of PARG should be assessed as a potential cause of clinical PARPi resistance. In this case, measurement of PARG activity should further improve clinical decision making for patients with tumours that lack homology-directed DNA repair.