Maintaining genome stability is crucial for all living cells and organisms. The repair of the highly cytotoxic DNA double-strand breaks is based on a variety of factors that modify chromatin structure, such as kinases and ubiquitin ligases. Our previous studies demonstrated that H2AK13Ub/H2AK15Ub promoted by RNF168 is a key event in this pathway. However, how the RNF168 pathway is functionally implicated in determining the repair pathway choice critical to genome maintenance by promoting either the error-prone NHEJ – via 53BP1 – or the error-free HR – via BRCA1 – is still missing. By using different experimental approaches, we found that phosphorylation of ubiquitin itself plays a key role in the fine modulation of DNA repair and the pathway choice in different phases of the cell cycle.