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Low temperature is a major adverse environmental factor that impairs petunia growth and development. To better understand the molecular mechanisms of cold stress adaptation of petunia plants, a quantitative proteomic analysis using iTRAQ technology was performed to detect the effects of cold stress on protein expression profiles in petunia seedlings which had been subjected to 2°C for 5d. Of the 2,430 proteins whose levels were quantitated, a total of 117 proteins were discovered to be differentially expressed under low temperature stress in comparison to unstressed controls. As an initial study, 44 proteins including well known and novel cold-responsive proteins were successfully annotated. By integrating the results of two independent Gene Ontology (GO) enrichment analyses, seven common GO terms were found of which oxidation-reduction process was the most notable for the cold-responsive proteins. By using the subcellular localization tool Plant-mPLoc predictor, as much as 40.2% of the cold-responsive protein group was found to be located within chloroplasts, suggesting that the chloroplast proteome is particularly affected by cold stress. Gene expression analyses of 11 cold-responsive proteins by real time PCR demonstrated that the mRNA levels were not strongly correlated with the respective protein levels. Further activity assay of anti-oxidative enzymes showed different alterations in cold treated petunia seedlings. Our investigation has highlighted the role of antioxidation mechanisms and also epigenetic factors in the regulation of cold stress responses. Our work has provided novel insights into the plant response to cold stress and should facilitate further studies regarding the molecular mechanisms which determine how plant cells cope with environmental perturbation.