Abstract

Barnyard grass ( Echinochloa crus‐galli ) is one of the world's most important weeds, and the evolution of herbicide resistance in this species threatens rice and maize production. Cytochrome P450–mediated metabolic herbicide detoxification is a prevalent resistance mechanism, yet its upstream regulatory control has remained elusive. This study uncovers an important regulatory module linking gibberellin (GA) signaling to herbicide detoxification. We identify the DOF‐family transcription factor DOF2 as an important regulator that directly activates herbicide detoxification genes, including  CYP81A21  and  CYP704A371  in  E. crus‐galli , and  CYP81A6  in rice. DOF2 is constitutively elevated in resistant  E. crus‐galli  and further induced by herbicide treatment. Overexpression of  DOF2  in  E. crus‐galli  or its ortholog in rice confers resistance, while knockout of the rice ortholog increases susceptibility. Mechanistically, the DELLA protein SLR1 interacts with DOF2 to repress its transcriptional activity, and GA signaling relieves this repression. We further show that herbicide treatment rapidly upregulates GA biosynthesis genes, leading to increased GA levels that trigger this pathway. Importantly, this GA–GID1/DELLA–DOF2–P450 axis operates in at least two Poaceae species, suggesting a conserved regulatory logic. These findings establish a direct mechanistic link between phytohormone signaling and metabolic herbicide resistance, with implications for weed resistance management and crop tolerance improvement. Plant hormone signaling modulates herbicide resistance in Echinochloa crus‐galli . The GA–DELLA–DOF2 cascade directly activates P450 detoxification genes in two Poaceae species, uncovering a core mechanism of metabolic herbicide resistance.