AbstractRoots exhibit hydrotropism in response to moisture gradients to avoid drought stress. Several proteins have been reported to regulate this process, with MIZU-KUSSEI 1 (MIZ1) being identified as a pivotal regulator. Although most studies on the regulatory mechanisms of root hydrotropism have focused on MIZ1, the molecular mechanisms of MIZ1 are poorly understood. Here, we report that MIZ1 plays an essential role in regulating cytokinin signal transduction by interacting with cytokinin receptors, ARABIDOPSIS HISTIDINE KINASEs (AHKs), in Arabidopsis (Arabidopsis thaliana). The miz1-2 mutant exhibited a decreased response to cytokinins, whereas overexpressors of MIZ1 showed an increased response to cytokinins. The expression levels of 2 Type-A Arabidopsis response regulators (ARRs) of cytokinins, ARR16 and ARR17, were downregulated, and their upregulation by cytokinins was substantially attenuated in miz1-2 compared with those in Col-0. Overexpression of MIZ1 partially rescued the decreased response of the ahk2-5 ahk3-7 double mutant to cytokinins. MIZ1 can physically interact with AHKs, as revealed by yeast 2-hybrid, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (co-IP) assays. Mutants of cytokinin signal transduction, such as ahk2-5 ahk3-7 ahk4-2 and arr3 arr4 arr5 arr6 arr8 arr9 arr16-C arr17-C, showed a greatly reduced hydrotropic response, similar to miz1-2. Additionally, MIZ1 also regulates the homeostasis of cytokinins by controlling the expression of genes encoding their biosynthetic and catabolic enzymes. Our results reveal the critical role of MIZ1 in regulating the cytokinin signaling response, which is essential for the root hydrotropic response.