Upland rice, an ecotype thriving in aerobic conditions and exhibiting drought tolerance, has an unclear genetic basis for its resistance. This study utilized a genome-wide association study (GWAS) combined with introgression line and transcriptomic analyses to identify *DROUGHT1* (*DROT1*), a gene encoding a COBRA-like protein that enhances drought resistance. *DROT1*, expressed in vascular bundles, is repressed by ERF3 and activated by ERF71, both drought-responsive transcription factors. It improves drought resistance by modulating cell wall structure, increasing cellulose content and maintaining cellulose crystallinity. A C-to-T single-nucleotide variation in the promoter boosts *DROT1* expression and drought resistance. The elite *DROT1* haplotype in upland rice possibly originated from wild rice (*O. rufipogon*) and could benefit upland rice breeding.
