AbstractThe nature of non‐target‐site herbicide resistance (NTSR) to imidazolinone (IMI) in HA425 sunflower (Helianthus annuus L.) has not yet been fully characterized but could be related to xenobiotic metabolism. The objective of this study was to evaluate the role of cytochrome P450 monooxygenases (P450s) and other detoxification‐related proteins in NTSR in sunflower. Two sunflower inbred lines were used: HA 425, which is IMI resistant (Imisun), and HA 89, which is IMI susceptible. The growth response to the IMI herbicide imazethapyr in combination with the P450 inhibitors 1‐aminobenzotriazole (ABT) or piperonyl butoxide (PBO) was evaluated in 15‐d‐old sunflower plantlets. Roots were collected, and label‐free quantitation (LFQ) proteomic analysis was carried out to characterize the NTSR mechanisms involved in the IMI resistance trait in sunflower. The increased phytotoxicity of imazethapyr observed in the resistant line when ABT or PBO were present agrees with the hypothesis that NTSR mechanisms may contribute to herbicide resistance in sunflower. The herbicide treatment also led to changes in the levels of biotic and abiotic stress‐related proteins, glutathione S‐transferases, and cytochrome P450s, among others. Plant growth and root protein expression response to IMI herbicides in sunflower would be a combination of stress‐related and detoxification mechanisms. Understanding the basis of NTSR becomes helpful to exploit this trait in sunflower crop and to develop xenobiotic‐resistant, soil‐remediating cultivars.