Recently, a novel type of abiotic stress caused by a prolongation of the light period - coined photoperiod stress - has been described in Arabidopsis. During the night after the prolongation of the light period, stress and cell death marker genes are induced. The next day, strongly stressed plants display a reduced photosynthetic efficiency and leaf cells eventually enter programmed cell death. The phytohormone cytokinin (CK) acts as a negative regulator of this photoperiod stress syndrome. In this study, we show that Arabidopsis wild-type plants increase the CK concentration in response to photoperiod stress. Analysis of cytokinin synthesis and transport mutants revealed that root-derived trans-zeatin (tZ)-type CKs protect against photoperiod stress. The CK signaling proteins ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 2 (AHP2), AHP3 and AHP5 and transcription factors ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), ARR10 and ARR12 are required for the protective activity of CK. Analysis of higher order B-type arr mutants suggested that a complex regulatory circuit exists in which the loss of ARR10 or ARR12 can rescue the arr2 phenotype. Together the results revealed the role of root-derived CK acting in the shoot through the two-component signaling system to protect from the negative consequences of strong photoperiod stress.