As the sole reproductive female in a honey bee (Apis mellifera) colony, the queen's health is critical to colony productivity and longevity. Beekeeping operations typically rely on the commercial mass production of queens for colony multiplication, which involves manipulating and isolating the queens by confining them in cages during early development. Using common queen-rearing techniques, this study shows that segregating newly eclosed queens from their worker attendants for 72 hours using queen protector cages has a significant impact on the total amount of gut bacteria carried by those queens compared to queens that have unrestricted access to attendants upon eclosion. Isolated virgin queens sampled immediately after isolation at 4 days post eclosure had significantly more bacteria and a less consistent microbiota composition than their non-isolated peers. Furthermore, this effect lasted into the mating life of queens, since mated queens that had been isolated after emergence and then sampled at 14 days post eclosure also had significantly more microbiota compared to non-isolated mated queens of the same age. The causes and potential impacts of this alteration are not clear and deserve further investigation. This study also verifies earlier findings that honey bee queens lack the core microbiome found within honey bee workers.