AtWRKY53 is an early factor in drought response and activated expression of AtWRKY53 regulates stomatal movement via reduction of H 2 O 2 content and promotion of starch metabolism in guard cells. Drought is one of the most serious environmental factors limiting the productivity of agricultural crops worldwide. However, the mechanisms underlying drought tolerance in plants remain unclear. AtWRKY53 belongs to the group III of WRKY transcription factors. In this study, we observed both the mRNA and protein products of this gene are rapidly induced under drought conditions. Phenotypic analysis showed AtWRKY53 overexpression lines were hypersensitive to drought stress compared with Col-0 plants. The results of stomatal movement assays and abscisic acid (ABA) content detection indicated that the impaired stomatal closure of 53OV lines was independent of ABA. Further analysis found that WRKY53 regulated stomatal movement via reducing the H2O2 content and promoting the starch metabolism in guard cells. The results of quantitative real-time reverse transcriptase PCR showed that the expression levels of CAT2, CAT3 and QQS were up-regulated in 53OV lines. Chromatin immunoprecipitation assays demonstrated that AtWRKY53 can directly bind to the QQS promoter sequences, thus led to increased starch metabolism. In summary, our results indicated that the activated expression of AtWRKY53 inhibited stomatal closure by reducing H2O2 content and facilitated stomatal opening by promoting starch degradation.