Stomata are composed of a pair of guard cells and a pore between them, and their density and positions are regulated by developmental and environmental signals. In a screen in which we overexpressed many genes coding for putative secretory proteins one by one in Arabidopsis, we identified a gene named STOMAGEN, which increases stomatal density when overexpressed. The STOMAGEN gene encodes a small peptide with a putative secretory signal sequence at its N-terminus and is expressed preferentially in mesophyll cells. This peptide belongs to the EPIDERMAL PATTERNING FACTOR (EPF) family of the cysteine-rich peptides superfamily. The mature form was a 45-amino-acid peptide (stomagen) with three intramolecular disulfide bonds. Stomagen treatment at very low concentrations, as low as 10 nM, increased the stomatal density of wild-type Arabidopsis plants. We propose that stomagen is a mesophyll-to-epidermis signaling molecule that positively regulates stomatal density. We also suggest that stomagen increases stomatal density by competing with negative regulators EPF1 and EPF2 for the receptor-like protein TOO MANY MOUTHS.