Cytokinin is a phytohormone involved in the regulation of diverse developmental and physiological processes in plants. Its potential for biotechnology and development of high-yield and more resilient plants has been recognized, but the molecular mechanisms behind its action are far from understood. In this report, the roots of barley seedling were explored as a new tool to reveal as yet unknown cytokinin-responsive proteins. Significant differences were reproducibly observed for 176 proteins, and at least some of the revealed cytokinin-responsive pathways were confirmed in metabolome analysis, including alterations in phenylpropanoid pathway, amino acid biosynthesis or ROS metabolism. Bioinformatics analysis indicated a significant overlap between cytokinin response and response to abiotic stress. This was confirmed by comparing proteome and metabolome profiles in response to drought, salinity or a period of temperature stress. The results illustrate complex abiotic stress response in the early development of model crop plant and confirm an extensive crosstalk between plant hormone cytokinin and response to temperature stimuli, water availability or salinity.