This study is a continuation of the innovative research of the impact of rearing of bee colonies simultaneously on standard- and small-cell combs on the traits of worker bees and bee colonies. Its aim was to compare the activities of proteases and their inhibitors in the hemolymph of workers reared in a small-cell comb (SMC) and a standard-cell comb (STC) in colonies kept simultaneously on standard- and small-cell combs. The width of comb cells in which workers are reared has a significant effect on the protein concentration and the proteolytic system in the hemolymph, which is reflected in the activities of proteases and their inhibitors. The protein concentrations in the 1-day-old workers were always higher (p ≤ 0.05) in the SMC than STC workers. The opposite was found in the older bee workers (aged 7, 14 and 21 d). The activities of proteases and their inhibitors in the 1-day-old workers were always higher (usually significantly at p ≤ 0.05) in STC than SMC workers, and opposite results were observed in the groups of the older workers (aged 7, 14 and 21 d). The differences between the workers from small-cell combs and those reared in standard-cell combs may be related to their different tasks. Workers reared in small-cell combs probably work as foragers outside the nest, whereas bees reared in standard-cell combs work in the nest. This hypothesis requires confirmation. To reduce the impact of accidental determinants on the results of single-season research on honeybees, it is advisable that such investigations should be conducted for several consecutive years.