Mating profoundly alters honey bee queen behavior and physiology. After mating, queens cease taking flights and remain in the hive, while vitellogenesis is initiated and egg-maturation is completed. There are also dramatic differences in the pheromone profiles of mated versus virgin queens, which lead to altered queen-worker interactions. Furthermore, honey bee queens mate multiply, with an average of approximately 12 males. While the effect of mating number on colony fitness have been considered in detail, it is unclear if there are direct effects of mating number on queen physiology or queen-worker interactions. We have used behavioral analyses, analytical chemistry, and functional genomics to determine the effects of mating number on honey bee queen pheromone profiles, queen physiology, and queen-worker interactions. Furthermore, we also examined the roles of the insemination volume and semen on producing these dramatic changes. Singly vs multiply inseminated queens have significantly different pheromone profiles, queen-worker interactions, vitellogenin levels, and brain gene expression levels. Insemination volume appears to be a major factor in producing these changes. These studies will begin to elucidate the effects of mating number at the level of queen physiology, gene expression and queen interactions with the workers, and will help characterize the molecular and physiological mechanisms underlying reproduction honey bee queens.