Honeybees can be evaluated to study their economic, developmental and immunity aspects. Apis mellifera has important organs such as hypopharyngeal gland, mandibular gland and brain in their heads. The hypopharyngeal glands and mandibular glands cooperate to produce royal jelly essential in feeding queen honeybees and broods. Moreover, the brain is an organ that regulates social behaviors of honeybees such as memory and learning. Therefore, this study aimed to detect differential head protein changes in one-day-old and nine-day-old worker honeybees. 153 repeatable protein spots were identified using Melanie software and statistically analyzed. Of the total protein spots, 27 responsive spots were identified using MALDI TOF-TOF mass spectrometry, of which 14 and 8 spots showed up and down regulation, respectively and five spots were expressed just in the head capsules of nine-day-old workers. All identified proteins were divided into 16 different groups involved in various growth-related processes as well as royal jelly augmentation. Nine proteins including KMT5B, TGIF2LX, Protein mono-ADP-ribosyltransferase PARP12, TCL1B4, maph-9, Vimentin-type intermediate filament-associated coiled-coil protein, HSP68, tetratricopeptide repeat domain 36 and POC1B were also identified and introduced for the first time in A. mellifera. This study provides valuable information on proteins involved in honeybee head growth and development as well as increasing royal jelly production.