The honeybee queen’s mandibular gland pheromones (QMP) are essential for the suppression of worker reproduction. Worker ovary activation is regulated by alternative splicing of a CP2-transcription factor named gemini. Since workers with activated ovaries also produce QMP in their mandibular glands, we tested whether alternative splicing of gemini also controls mandibular gland pheromone biosynthesis in workers using RNA interference. Altering the splice pattern of gemini resulted in enhanced levels of the queen-specific components of the mandibular gland pheromone in queenless honeybee workers, suggesting that gemini functions as a pleiotropic regulatory switch influencing both ovary activation and resulting in QMP synthesis in workers. Because the QMP produced by these workers suppresses ovary activation in other workers, gemini seems to be a key regulatory gene affecting reproductive hierarchies among workers in queenless colonies.