Cold tolerance is the main feature of life activity in Prunus persica, which directly affects the introduction and breeding for resistance. However, the molecular mechanisms underlying this process remain unknown. Therefore, isobaric tag for relative and absolute quantification (iTRAQ)-based comparative proteomic analysis was conducted to identify cold-responsive proteins in cold-tolerant peach cultivar (CT) and cold-sensitive peach cultivar (CS) in response to cold stress. This study examined 2,575 different proteins, among which, 322 and 399 showed significantly differential accumulation within CS and CT peach cultivars, respectively, following cold stress for 48 h. As suggested by bioinformatic analyses, compared with CS, CT potentially displayed a relationship with the greater amount of energy generated from carbon metabolism, starch and sucrose metabolism, and phenylpropanoid biosynthesis to resist cold stress. Peroxidase, flavonoid, carbonic anhydrase, and harpin proteins were more abundant in CT, which suggested that they might play roles in mitigating the cold resistance-induced damage. Besides, a variety of strategies were utilized to respond to cold stress in CT and CS, due to the different pathogenesis-related (PR) proteins. In addition, real-time PCR (RT-PCR) indicated that five candidate protein-encoding genes (M5WUFO, M5W9D5, H9EBJ3, Q5QIB3, and M5W6Z4) were highly expressed, which were consistent with iTRAQ data in CT. Taken together, our results suggest that five genes are involved in modulating the cold stress mechanisms of CT. Our study provides useful information for understanding the underlying mechanisms of cold tolerance and identifying the candidate resistant proteins in peach, which possibly facilitates to breed peach cultivars with enhanced cold tolerance.