Background and Aims <p>As a globally essential food crop, the flowering time of <i>Zea</i> <i>mays</i> is fundamental to its reproductive development, yield formation, and adaptation to various agro-climatic conditions. Concurrently, drought represents a critical environmental factor that significantly limits maize yields worldwide. The AP2/ERF transcription factor superfamily is widely recognized for its crucial roles in regulating both developmental processes, such as seed development, plant height, flowering, and plant responses to abiotic stresses including drought, high temperature, low temperature. The Dehydration Responsive Element Binding protein (DREB) subfamily is a member of the AP2/ERF transcription factor family. In this study, we analyzed the function of the <i>ZmDREB53</i> gene and elucidated its dual roles in the regulation of flowering and drought tolerance. Therefore, this study aims to systematically investigate the function of <i>ZmDREB53</i> in two key aspects: the control of flowering time and the enhancement of drought tolerance, thereby addressing the significant knowledge gap surrounding this gene.</p> Methods <p>In this study, a multi-layered analysis of phenotypic and physiological indices was performed on transgenic <i>Arabidopsis thaliana</i> plants, with systematic evaluation of their performance in growth, physiological, and yield-related traits. For growth and developmental traits, the number of flower buds, petal length, and silique length were determined in Wild-Type (WT) and overexpressing <i>A. thaliana</i> at the flowering stage. On 1/2 MS medium under mannitol-simulated drought stress, seed germination rates of the two <i>A. thaliana</i> lines were recorded after 6&#xa0;days of treatment; primary root length and root hair density were measured following 18&#xa0;days of treatment. Under normal growth conditions and after 20&#xa0;days of natural drought stress, superoxide anion (O₂^⁻) was detected via nitroblue tetrazolium (NBT) staining. Combined with 3,3'-diaminobenzidine (DAB) staining, reactive oxygen species (ROS) accumulation in transgenic and WT was quantitatively analyzed. Concomitantly, the content of hydrogen peroxide (H₂O₂), activities of antioxidant enzymes [peroxidase (POD),&#xa0;catalase (CAT) and superoxide dismutase (SOD)], and level of malondialdehyde (MDA)—a core marker of lipid peroxidation—were determined. Furthermore. Furthermore, the expression profiles of key genes in the flowering pathway were analyzed via RT-qPCR, providing necessary molecular evidence.</p> Results <p>Our analysis revealed that <i>ZmDREB53</i> exhibits predominant expression in maize seeds and is localized within the nucleus. Transgenic <i>A. thaliana</i> flowered 7–10&#xa0;days earlier than WT plants, with no significant changes in flower or pod traits. Additionally, the transgenic plants exhibited stronger ROS scavenging ability and higher drought resistance compared to WT plants.</p> Conclusion <p>Overall, <i>ZmDREB53</i> functions as a positive regulator of flowering time in <i>A. thaliana</i> and contributes to enhanced drought tolerance. These findings offer valuable insights for elucidating the mechanisms of growth regulation in maize and provide a genetic resource for breeding improved varieties with stronger stress resistance.</p> Graphical Abstract <p></p>