DNA double-strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle1. In interphase, DSBs are mainly repaired by non-homologous end joining and homologous recombination2. However, these pathways are completely inhibited in mitosis3–5, leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta6 (Polθ) repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair facto...