There are the two major pathways responsible for the repair of DNA double-strand breaks (DSBs): non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ operates throughout the cell-cycle, while HR is primarily active in the S/G2 phases suggesting that there are cell cycle-specific mechanisms that regulate the balance between NHEJ and HR. Here we reported that CDK2 could phosphorylate RNF4 on T26 and T112 and enhance RNF4 E3 ligase activity, which is important for MDC1 degradation and proper HR repair during S phase. Mutation of the RNF4 phosphorylation sites results in MDC1 stabilization, which in turn compromised HR during S-phase. These results suggest that in addition to drive cell cycle progression, CDK also targets RNF4, which is involved in the regulatory network of DSBs repair.