cold is one of the most significant abiotic stresses which restrict crop growth and productivity worldwide. In order to investigate how spring barley (Hordeum vulgare L.) seedlings adapt to short-term periods of low temperature, the present study explored proteomic changes in leaves. Cold stress at 4 °C was applied to barley seedlings for 48 hours; third leaves were harvested and compared with seedlings grown in normal conditions (25° C). The proteomic analysis was conducted by two-dimensional electrophoresis (2-DE) and the Coomassie blue staining procedure. Fifteen reproducible protein spots showing a significant difference between the control condition and cold stress were identified; 10 of the spots demonstrated an increase in expression while 5 spots showed a decrease under 4 °C cold stress for 48 hours. By applying MALDI-TOF analysis, 7 spots were identified. These responsive proteins were involved in the Calvin cycle, photosynthetic electron transport, light reaction, and signal transduction. The upregulation of proteins involved in the regulation of the chloroplast system, the integrity of chloroplasts, energy metabolism, antioxidant defense, and photosynthesis has probably acclimatized the plant to cold stress. These findings indicate that there was greater cold stress affecting photosynthesis in spring barley and it is of crucial importance to maintain the efficiency of photosynthesis under cold stress.