The application of uranium (U) in the nuclear energy and defense industry has driven U mining activities, leading to subsequent U contamination. Understanding the toxicity and detoxification mechanism of U in plants is crucial for enhancing the efficiency of phytoremediation efforts in U-contaminated soils. The present study investigated the toxicity of uranium (U) in radish and its impact on physiological and molecular responses. The application of U (5-25 μM) for 3 days significantly inhibited the elongation of radish lateral roots, and the lateral root length decreased by 35.6%-60.7% compared with the control. Under U stress, radish root tip meristem cells suffered DNA damage, fortunately the cells remained viable. To repair damaged DNA, the expression of genes involved in DNA repair (e.g. RAD2, XPC, BLM) was up-regulated, and the expression of genes involved in cell cycle was down-regulated (e.g. CYCB, CDKB). Under U stress, the expression of respiratory burst oxidase homologs (RBOHs) genes in radish roots up-regulated, which caused ROS burst, and then enhanced autophagy by promoting the expression of autophagy related genes (ATGs). Simultaneously, the glutathione (GSH) content increased, and the gene expression levels and activities of antioxidant enzymes (e.g. catalase) were increased, which enhanced the antioxidant capacity of root cells. Moreover, ubiquitin-proteasome system (UPS) (e.g. E3 ligase genes NEDD4) was involved in the activation of DNA repair, GSH synthesis and autophagy. In summary, DNA repair, autophagy, and antioxidant systems were activated in radish roots, which promoted the survival of apical meristem cells under U stress.