Pollen is the primary source of dietary protein for honey bees. It also includes complex polysaccharides in its outer coat, which are largely indigestible by bees but can be metabolized by bacterial species within the gut microbiota. During periods of reduced availability of floral pollen, supplemental protein sources are frequently provided to managed honey bee colonies. The crude proteins in these supplemental feeds are typically byproducts from food manufacturing processes and are rarely derived from pollen. Our experiments on the impact of different diets showed that a simplified pollen-free diet formulated to resemble the macronutrient profile of a monofloral pollen source resulted in larger microbial communities with reduced diversity, reduced evenness, and reduced levels of potentially beneficial hive-associated bacteria. Furthermore, the pollen-free diet sharply reduced the expression of genes central to honey bee development. In subsequent experiments, we showed that these shifts in gene expression may be linked to colonization by the gut microbiome. Lastly, we demonstrated that for bees inoculated with a defined gut microbiota, those raised on an artificial diet were less able to suppress infection from a bacterial pathogen than those that were fed natural pollen. Our findings demonstrate that a pollen-free diet significantly impacts the gut microbiota and gene expression of honey bees, indicating the importance of natural pollen as a primary protein source.