Updated project metadata. Ki-67 molecular functions and properties have remained quite obscure for a long time, despite its importance as a major proliferation marker in clinic. Only recently it has been shown that Ki-67 has a major role in the formation of the mitotic chromosome periphery compartment, and it is a protein phosphatase 1 (PP1) binding protein. Understanding Ki-67 functions at different stages of the cell cycle has been so far hindered by the fact that the different phenotypes observed in cells upon Ki-67 depletion could be the outcome of secondary effects. Here, by using Auxin-inducible-degron (AID) system, we show that Ki-67 degradation at the G1/S boundary causes the disassembly of the replication machinery and leads to DNA damage. This triggers the interferon response and causes replication delay. Our results support the model whereby Ki-67 contributes to replication forks protection, and it is important for timely DNA replication and genome maintenance.