Cold is one of the most significant abiotic stresses that restrict crop growth and productivity worldwide. In order to investigate how spring barley (Aths cultivar) adapts to short-term cold stress, the present study attempted to explore proteomic, physiological, and biochemical changes that occur in leaves. Barley seedlings were exposed to low temperature (4 C) for 48 hours, and third leaves were harvested and compared with plants grown in normal conditions (25 C). In cold-stressed plants, results indicated a significant increase in hydrogen peroxide content, associated with a highly significant rise of lipid peroxidation (as demonstrated by MDA measurements) and catalase activity; the latter represents one of the oxidative stress resistance strategies adopted to promote cell survival. Cold stress also caused a significant reduction in chlorophyll b (chlb) content with a parallel increase in the chla/chlb ratio, which is probably related to defense mechanisms against cold-induced production of reactive oxygen species. On the contrary, other physiological and biochemical traits [namely, the membrane stability index (MSI), peroxidase activity, electron leakage, carotenoid and chla content] showed no statistically significant differences. The proteomic analysis revealed fifteen statistically significant cold-modulated spots, seven of which were successfully identified by LC-MS/MS. These responsive proteins are related to the Calvin cycle, photosynthetic electron transport, light reactions, and signal transduction. An increase in abundance of proteins involved in the regulation of the chloroplast system probably reflects plant acclimation to cold, thus confirming that …