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Background As one of the most important vegetable crops, pepper has rich nutritional value and high economic value. Increasing heat stress due to the global warming has a negative impact on the growth and yield of pepper. Methods To understand the heat stress response mechanism of pepper, an iTRAQ-based quantitative proteomic analysis was employed to identify possible heat-responsive proteins and metabolic pathways in 17CL30 and 05S180 pepper seedlings under heat stress. Result In the present study, we investigated the changes of phenotype, physiology, and proteome in heat-tolerant (17CL30) and heat-sensitive (05S180) pepper cultivars in response to heat stress. Phenotypic and physiological changes showed that 17CL30 had a stronger ability to resist heat stress compared with 05S180. In proteomic analysis, a total of 3,874 proteins were identified, and 1,591 proteins were considered to participate in the process of heat stress response. According to bioinformatic analysis of heat-responsive proteins, the heat tolerance of 17CL30 might be related to a higher ROS scavenging, photosynthesis, signal transduction, carbohydrate metabolism, and stress defense, compared with 05S180.