The favorable responses of crop species to low-dose gamma irradiation can help to develop cultivars with increased productivity and improved stress tolerance. In the present study, we tried to reveal the candidate metabolites involved in growth stimulation of barley seedlings after applying low-dose gamma-radiation (Co-60) to seeds. Stimulating doses (5-20 Gy) provided a significant increase in shoot length and biomass, while relatively high dose of 100 Gy led to significant inhibition of growth. Gas chromatography-mass spectrometry metabolomic analysis uncovered several compounds that may take part in radiation hormesis establishment in irradiated plants. This includes molecules involved in nitrogen redistribution (arginine, glutamine, asparagine, and gamma-aminobutyric acid) and stress-responsive metabolites, such as ascorbate, myo-inositol and its derivates, and free amino acids (l-serine, beta-alanine, pipecolate, and GABA). These results contribute to the understanding of the molecular mechanisms of hormesis phenomenon.