Cytokinins (CKs) regulate many developmental processes and environmental stress responses in plants. In this study, our data provide evidence that CK negatively regulates Arabidopsis selenium (Se) stress response. CK-deficient plant ipt1 3 5 7 exhibited enhanced Se tolerance which was abolished by exogenous benzylaminopurine (BA) application, while CK- receptor -deficient mutants ahk2 and ahk3 were sensitive to Se stress. Further investigation suggested that CK regulated Se tolerance of ipt1 3 5 7 through reduction of Se uptake and activation of metabolism detoxification, which had significantly lower transcriptions of high-affinity transporters PHT1;1, PHT1;8, PHT1;9 and the higher transcription of selenocysteine methyltransferase (SMT) respectively. Moreover, Se tolerance of ipt1 3 5 7 was associated with the enhanced antioxidant levels which had the higher catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX) activities as well as the higher glutathione (GSH) content. On the other hand, loss-of-function mutations in single CK receptor genes could increase Se uptake and reactive oxygen species (ROS) accumulation, which caused Se sensitivity in ahk2 and ahk3 mutants. Taken together, these findings provide new insights to the role of CK in Se stress response in Arabidopsis.