Understanding the mechanisms that endow a somatic cell with the ability to differentiate into a somatic embryo, which could result in numerous biotechnological applications, is still a challenge. The objective of this work was to identify some of the molecular and physiological mechanisms responsible for the acquisition of embryogenic competence during somatic embryogenesis in Carica papaya L. We performed a broad characterization of embryogenic (ECs) and nonembryogenic calli (NECs) of C. papaya using global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide contents. ECs and NECs presented remarkable differences in anatomical and histochemical characteristics. ECs showed greater reactivity for the presence of proteins and neutral polysaccharides. Our results demonstrate the role of mitochondrial metabolism in the embryogenic competence of C. papaya calli. Greater participation of alternative oxidase (AOX) enzymes in respiration, as well as stronger accumulation of mitochondrial stress response proteins, was observed in ECs. In addition, ECs showed a greater abundance of proteins related to oxidative phosphorylation and higher total respiration (TR). Auxin-responsive Gretchen Hagen 3 (GH3) family proteins may play an important role in decreasing the contents of free 2,4-dichlorophenoxyacetic acid (2,4-D) in ECs. The accumulation of stress response proteins among total proteins was observed in ECs. ECs also showed higher endogenous hydrogen peroxide (H2O2) contents. H2O2 is a promising molecule for further investigation in differentiation protocols for C. papaya somatic embryos.