The aim of the study was to determine the effect of cannabidiol extract (CBD) on the activity of the proteolytic system and the concentrations of total proteins on the cuticular surface of worker bees. Marked one-day-old workers were introduced into established colonies, which were divided into three groups (two hives each): CSy (CBD in sugar syrup), CSt (CBD-soaked strip), and C (control, pure syrup). Every seven days, the bioactive cuticular layer was washed off from the marked bees for biochemical analysis. Total protein concentrations increased with worker age in C, CSy, and CSt, and were highest from day 7 in group C. Protease activities increased until day 21/28 of worker life in all groups. From day 21 for acidic proteases, day 28 for neutral proteases, and day 14 for alkaline proteases, the activities were highest in CSy. The activities of neutral and alkaline protease inhibitors decreased with bee age and were always higher in CSy and CSt than in C, regardless of worker age. CBD favorably modulates the proteolytic system on the cuticle of honeybees