Honeybees (Apis mellifera Linnaeus, 1758) are managed pollinators in anthropized landscapes but suffer adverse physiological effects from urbanization due to increased pollution, higher temperatures and a loss of habitat quality. Previous studies in various animal taxa have shown how responses of digestive enzymes, such as Aminopeptidase N (APN), can indicate stress conditions and thus be used to measure the harmfulness of anthropogenic disturbance. However, no studies have focused on bees. Here, we sampled honeybee foragers along an urbanization gradient in the Metropolitan City of Milan (Italy) and measured the APN activity. After briefly characterizing the midgut APN activity under different pH and temperature conditions, we found that APN activity was lower at urban sites with higher temperatures (Urban Heat Island (UHI) effect). Furthermore, an increasing proportion of meadows (semi-natural flowered areas) and a decreasing proportion of urban parks (managed urban green areas)—both higher in less urbanized sites—were associated with higher APN activity. Our results suggest that severe urban conditions may cause a reduction in APN activity, but that the UHI effect alone is not directly involved. Although the actual urbanization-related factors driving our results remain unclear, we suggest that impoverishment of food sources may play a role. As aminopeptidases are involved in pollen digestion, our results may indicate a possible impairment of the digestive capacity of honeybees in highly urbanized areas.