Cytokinin is an essential phytohormone in plant growth and development. In Arabidopsis, cytokinin signalling is mediated by a phosphorelay that sequentially transfers phosphoryl groups from the cytokinin receptors to histidine phosphotransfer proteins (AHPs) and response regulators (ARRs). However, little is known about the regulatory mechanism of the phosphorelay. Here, we show that nitric oxide negatively regulates cytokinin signalling by inhibiting the phosphorelay activity through S-nitrosylation. S-nitrosylation of AHP1 at Cys 115 represses its phosphorylation and subsequent transfer of the phosphoryl group to ARR1. A non-nitrosylatable mutation of AHP1 renders the mutant protein insensitive to nitric oxide in repressing its phosphorylation, and partially relieves the inhibitory effect of nitric oxide on the cytokinin response. Conversely, a nitrosomimetic mutation of AHP1 causes reduced phosphorylation of AHP1 and ARR1, thereby resulting in a compromised cytokinin response. These findings illustrate a mechanism by which redox signalling and cytokinin signalling coordinate plant growth and development.