In plants, the cytokinin metabolic processes, including cytokinin biosynthesis, interconversion, inactivation, and degradation, play critical roles in the regulation of cytokinin homeostasis and plant development. Purine metabolic enzymes have been implied to catalyze the cytokinin interconversion in previous works. In this study, we report that Adenine Phosphoribosyl Transferase 1 (APT1) is the causal gene of the high-dose cytokinin-resistant mutants. APT1 catalyzes the cytokinin conversion from free bases to nucleotides, and is functionally predominant among the five members of the Arabidopsis Adenine Phosphoribosyl Transferase family. Loss of APT1 activity in plants leads to excess accumulation of cytokinin bases, thus evoking myriad cytokinin-regulated responses, such as delayed leaf senescence, anthocyanin accumulation, and downstream gene expression. Thus, our study defines APT1 as a key metabolic enzyme participating in the cytokinin inactivation by phosphoribosylation.