Main conclusion <p>Melatonin integrates multiple biological pathways to enhance rice drought tolerance with cooperation of WRKY, bHLH, ERF, MYB and NAC transcription factors, and crosstalk of salicylic acid, ethylene and auxin.</p> Abstract <p>Drought is one of the primary environmental threats, and detrimentally affects plant growth and development, impeding crop yield and food quality worldwide. Melatonin has recently emerged as a multifunctional biomolecule with promising aspects in plant stress tolerance. However, the way in which melatonin improves drought tolerance in rice has not been investigated systematically. Here, we demonstrate that melatonin minimized drought effects on rice, resulting in improved germination rate and growth performance. Application of exogenous 200 and 400 µM melatonin can significantly inhibit the accumulation of reactive oxygen species in rice, by enhancing the activity of ROS scavenging enzymes. The accumulation of osmolytes was also stimulated by melatonin to endure with a better preservation of leaf water status in drought-stressed rice. Genome-wide expression profiling by RNA sequencing reveals an increase of oxidoreductase activity, iron ion binding, hydrolase activity, cell wall biogenesis, root development, while a decrease of nitrogen compound metabolism and cellular biosynthesis. Furthermore, melatonin could modulate rice drought response through the cooperation of WRKY, bHLH, ERF, MYB and NAC transcription factors, and modulates salicylic acid, ethylene and auxin pathways. Our finding provides new insights into melatonin-mediated drought tolerance in rice, and facilitates the rational applications in stress management for agricultural, horticultural, and floricultural plants.</p>