Honey bee queens are exceptionally promiscuous. Early in life, queens perform one to five nuptial flights, mating with up to 44 drones. Many studies have documented potential benefits of multiple mating. In contrast, potential costs of polyandry and the sensitivity of queens to such costs have largely been ignored because they are difficult to address experimentally. To consider one aspect of mating costs to queens, the difficulty of flight, we compared flight behavior and success among a group of control queens and two experimental groups of queens that carried lead weights of two different sizes. For each queen, we assessed the number and duration of all flights and, after egg-laying commenced, the amount of stored sperm and the number of mates in terms of the offspring’s patrilineal genetic diversity. Added weights quantitatively decreased the number of flights, the mean duration of flights and consequently the total time spent flying. Mating success in terms of sperm quantity and patrilines detected among the queens’ offspring was also negatively impacted by the experimental manipulation. Thus, it can be concluded that the flight effort of honey bee queens during their mating period is adjusted in response to an experimentally increased cost of flying with multiple consequences for their mating success. Our results suggest that queen behavior is flexible and mating costs deserve more attention to explain the extreme polyandry in honey bees.