Sweet cherry (Prunus avium L.) is a delicious nutrient-rich fruit widely cultivated in countries such as China, America, Chile, and Italy. However, the yield often drops severely due to the frequently-abnormal fruitlet abscission, and few studies on the metabolism during its ripening process at the proteomic level have been executed so far. To get a better understanding regarding the sweet cherry abscission mechanism, proteomic analysis between the abscising carpopodium and non-abscising carpopodium of sweet cherry was accomplished using a newly developed Liquid chromatography-mass spectrometry/mass spectrometry with Tandem Mass Tag (TMT-LC-MS/MS) methodology. The embryo viability experiments showed that the vigor of the abscission embryos was significantly lower than that of retention embryo. The activity of cell wall degrading enzymes in abscising carpopodium was significantly higher than that in non-abscising carpopodium. The anatomy results suggested that cells in the abscission zone were small and separated. In total, 6280 proteins were identified, among which 5681 were quantified. It has been observed that differentially accumulated proteins (DAPs) influenced several biological functions and various subcellular localizations. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that plenty of metabolic pathways were notably enriched, particularly those involved in phytohormone biosynthesis, cell wall metabolism, and cytoskeletal metabolism, including 1-aminocyclopropane-1-carboxylate oxidase proteins which promote ethylene synthesis, and proteins promoting cell wall degradation, such as endoglucanases, pectinase, and polygalacturonase. Differential expression of proteins concerning phytohormone biosynthesis might activate the shedding regulation signals. Up-regulation of several cell wall degradation-related proteins possibly regulated the shedding of plant organs. Variations of the phytohormone biosynthesis and cell wall degradation-related proteins were explored during the abscission process. Furthermore, changes in cytoskeleton-associated proteins might contribute to the abscission of carpopodium. The current work represented the first study using comparative proteomics between abscising carpopodium and non-abscising carpopodium. These results indicated that embryo abortion might lead to phytohormone synthesis disorder, which effected signal transduction pathways, and hereby controlled genes involved in cell wall degradation and then caused the abscission of fruitlet. Overall, our data may give an intrinsic explanation of the variations in metabolism during the abscission of carpopodium.