Maize production is threatened by drought stress worldwide.Identification of the genetic components underlying drought tolerance in maize is of great importance.Here,we report a genome-wide association study (GWAS) of maize drought tolerance at the seedling stage that identified 83 genetic variations which were resolved to 42 candidate genes.The peak signal of GWAS uncovered that the natural variation in ZmVPP1,encoding a vacuolar-type H+-pyrophosphatase,most significantly contributes to the trait.Further analysis found that a 366-bp insertion in the promoter,containing three MYB cis-elements,confers drought-inducible expression of ZmVPP1 in drought-tolerant genotypes.Introgression the tolerant allele of ZmVPP1 into drought sensitive genetic background greatly improved seedling drought stress tolerance.Transgenic maize with enhanced ZmVPP1 expression exhibits improved drought tolerance,most likely due to enhanced photosynthetic efficiency and root development.Taken together,this research provides important genetic insights into the natural variation of maize drought tolerance.The identified loci/genes can serve as direct targets for both genetic engineering and selection for the trait improvement of maize.