Peroxisomes are ubiquitous organelles from eukaryotic cells characterized by an active nitro-oxidative metabolism. They have a relevant metabolic plasticity depending on the organism, tissue, developmental stage, or physiological/stress/environmental conditions. Our knowledge of peroxisomal metabolism from fruits is very limited but its proteome is even less known. Using sweet pepper (Capsicum annuumL.) fruits at two ripening stages (immature green and ripe red), it was analyzed the proteomic peroxisomal composition by quantitative isobaric tags for relative and absolute quantitation (iTRAQ)-based protein profiling. For this aim, it was accomplished a comparative analysis of the pepper fruit whole proteome obtained by iTRAQ versus the identified peroxisomal protein profile fromArabidopsis thaliana. This allowed identifying 57 peroxisomal proteins. Among these proteins, 49 were located in the peroxisomal matrix, 36 proteins had a peroxisomal targeting signal type 1 (PTS1), 8 had a PTS type 2, 5 lacked this type of peptide signal, and 8 proteins were associated with the membrane of this organelle. Furthermore, 34 proteins showed significant differences during the ripening of the fruits, 19 being overexpressed and 15 repressed. Based on previous biochemical studies using purified peroxisomes from pepper fruits, it could be said that some of the identified peroxisomal proteins were corroborated as part of the pepper fruit antioxidant metabolism (catalase, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductaseglutathione reductase, 6-phosphogluconate dehydrogenase and NADP-isocitrate dehydrogenase), the β-oxidation pathway (acyl-coenzyme A oxidase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase), while other identified proteins could be considered “new” or “unexpected” in fruit peroxisomes like urate oxidase (UO), sulfite oxidase (SO), 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (METE1), 12-oxophytodienoate reductase 3 (OPR3) or 4-coumarate-CoA ligase (4CL), which participate in different metabolic pathways such as purine, sulfur,L-methionine, jasmonic acid (JA) or phenylpropanoid metabolisms. In summary, the present data provide new insights into the complex metabolic machinery of peroxisomes in fruit and open new windows of research into the peroxisomal functions during fruit ripening.