Keratinocytes, the major cell types of the epidermis, proliferate and migrate during wound healing to restore the epithelial barrier. Royal jelly is a traditional remedy used in wound repair. Our previous study found that the mixture of major royal jelly protein (MRJP) 2, 3 and 7 exhibited in vitro wound healing-promoting effects; however, the exact functional constituents and the associated underlying mechanisms of action are still largely unknown. In this study, a partial fragment of MRJP3 was recombinantly expressed as a fusion protein MRJP3-C113-Fc which promoted wound healing in vitro and in vivo. By employing protein inhibitors and immunoblots technology, it was initially found that the wound-repairing mechanisms of MRJP3-C113-Fc were correlated to the activation of EGFR/AKT/mTOR signaling pathway in keratinocytes (HaCaT cells). LC-MS/MS-based proteomic analysis demonstrated that the proteins present in the MRJP3-C113-Fc-treated HaCaT cells were different from the untreated ones, in which rhomboid 5 homolog 2 (RHBDF2) might be the potential regulator for the EGFR/AKT/mTOR signaling pathway. Silencing of RHBDF2 diminished the pro-proliferative and -migratory effectiveness of MRJP3-C113-Fc on HaCaT cells, as well as the phosphorylation of EGFR/AKT/mTOR, suggesting that the wound healing-promoting efficacy was attributable to the RHBDF2-mediated activation of EGFR/AKT/mTOR signaling pathway. Hence, this study is the first to discover the single fragment of MRJPs possessing pro-healing properties, and also the first to disclose the regulatory role of RHBDF2 in EGFR/AKT/mTOR signaling pathway-modulated wound healing. It will facilitate the development of MRJP3-C113-based therapeutic agent for skin wounds, and provide a novel target for treating cutaneous trauma.