Grafting is a technology widely used in agriculture and biological research. During a hetero-graftingprocess, cutting wound and hetero-grafting per se are the two main stimuli invoking cellular and whole-plant responses. Despite the importance of dissecting the individual effects of the two in adequatelyinterpreting experimental data, no currently published data are dedicated to comparing the effects ofhetero-grafting per se and grafting wound. In this study, isobaric tags for relative and absolute quan-titation (iTRAQ) technique-based proteomic analyses were conducted between various scion-rootstockcombinations of bottle gourd. Our results demonstrated that effects of grafting wound and hetero-graftingper se were readily distinguishable at the proteome level. Grafting wound affected the proteomes differ-entially at the graft union and its upper vicinity. Its effects were also likely related to the stress resistancelevel of each genotype. The effects of hetero-grafting per se on proteome were markedly different betweengraft union and the upper vicinity. This study provides an explicit evidence for the effectiveness of thecommonly used methods of eliminating the effects of grafting wound in a hetero-grafting system bysetting up a homo-grafted control. The role of hydrogen peroxide as a signal molecule in hetero-graftingsystems is suggested.