Recent studies have pointed to PARP1 trapping as a key determinant of the anticancer effects of PARP1 inhibitors (PARPi). We identified RNF114, as a PARylation-dependent, E3 ubiquitin ligase involved in DNA damage response. Upon sensing genotoxicity, RNF114 was recruited, in a PAR-dependent manner, to DNA lesions, where it targeted PARP1 for degradation. The blockade of this pathway interfered with the removal of PARP1 from DNA lesions, leading to profound PARP1 trapping. We showed that a natural product, nimbolide, inhibited the E3 ligase activity of RNF114 and thus caused PARP1 trapping. However, unlike conventional PARPi, nimbolide treatment induced the trapping of both PARP1 and PARylation-dependent DNA repair factors. Nimbolide showed synthetic lethality with BRCA mutations, and it overcame intrinsic and acquired resistance to PARPi, both in vitro and in vivo. These results point to the exciting possibility of targeting the RNF114-PARP1 pathway for the treatment of homologous recombination-deficient cancers.