Plant miRNA regulates multiple developmental and physiological processes, including drought responses. We found that the accumulation of Sly-miR169 in tomato (Solanum lycopersicum) was induced by drought stress. Consequently, Sly-miR169 targets, namely, three nuclear factor Y subunit genes (SlNF-YA1/2/3) and one multidrug resistance-associated protein gene (SlMRP1), were significantly down-regulated by drought stress. Constitutive over-expression of a miR169 family member, Sly-miR169c, in tomato plant can efficiently down-regulate the transcripts of the target genes. Compared with non-transgenic plants, transgenic plants over-expressing Sly-miR169c displayed reduced stomatal opening, decreased transpiration rate, lowered leaf water loss, and enhanced drought tolerance. Our study is the first to provide evidence that the Sly-miR169c negatively regulates stomatal movement in tomato drought responses.