Genome integrity is important for cell growth, development and proliferation. The E3 ligase RAD18 plays a vital role in the DNA damage response (DDR) to maintain genome integrity. Recent studies reveal that RAD18 has non-ubiquitinated and mono-ubiquitinated form in normal cells. However, whether RAD18 undergoes other post-translational modification remains to be investigated. Here we show that RAD18 is a target of NEDD8, an ubiquitin-like protein. In response to hydrogen peroxide (H2O2)-induced oxidative stress, RAD18 NEDDylation increases significantly, while its ubiquitination decreases. Moreover, NEDD8 overexpression or deNEDDylase NEDP1 deletion further antagonizes RAD18 ubiquitination. In addition, treatment with MLN4924, an inhibitor of NEDD8-activating Enzyme, reduces the interaction between PCNA and RAD18, which blocks the localization of RADI8 to form foci, and thus inhibiting polymerase eta recruitment after oxidative stress. Together, our study demonstrates that RAD18 NEDDylation regulates its localization and involves in the DDR pathway by modulating RAD18 ubiquitination. (C) 2018 Elsevier Inc. All rights reserved.