Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H ₂O ₂ production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H ₂O ₂, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H ₂O ₂ production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H ₂O ₂ production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect. Significance Increasing evidence supports the role of chronic oxidative stress in late radiation-induced effects, including malignancy and genetic instability. To date, elevated levels of reactive oxygen species (ROS) have been considered a cause of persistent instability, but until now the mechanism(s) underlying the perpetuation of ROS generation in irradiated cells and their progeny was undetermined. Cells can produce ROS through activation and/or induction of NADPH oxidases. The present investigation identifies the DUOX1-based NADPH oxidase as a ROS-generating system induced after irradiation, causing delayed DNA breakage. Overexpression of DUOX1 in radio-induced thyroid tumors suggests that DUOX1 may contribute to a chronic oxidative stress promoting genomic instability and tumorigenesis.