Significance Stomata are widespread in aerial part of plants as passages exchanging gas and water with environment. Therefore, stomata are crucial for photosynthesis as well as global carbon and water circulation. Auxin, as the first identified phytohormone, participates in many aspects of plant growth and development, but whether auxin regulates stomatal development is unknown. This study establishes that auxin negatively regulates stomatal development through MONOPTEROS (MP) repression of mobile peptide gene STOMAGEN expression in mesophyll cells, which is mediated by direct binding of MP to auxin response elements in the STOMAGEN promoter. This study advances our knowledge about the roles of auxin and the versatile regulator MP in plant growth and development.