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[Display omitted] • The 7−OH MTX was the main culprit for MTX nephrotoxicity. • PERK/CHOP mediated ER stress was screened as key pathway by RNA-seq analysis. • Nobiletin could ameliorate the nephrotoxicity of 7−OH MTX through PERK/CHOP pathway. • Nobiletin could maintain Ca2+ homeostasis and reduce the production of ROS. Drug-induced nephrotoxicity is a frequent adverse event that contributes to acute kidney injury with tubular and/or glomerular lesions. Methotrexate (MTX) is a folate analog used against a myriad of malignancies and autoimmune diseases. Unfortunately, ambiguous renal toxicology limits its safe clinical usage. Based on our previous studies, 7−OH MTX as an overlooked oxidative metabolite of MTX was proposed to be the main culprit responsible for nephrotoxicity, while nobiletin, a naturally occurring polymethoxylated flavonoid screened from our prepared total phenolic extracts of Citrus aurantium L. (TPE-CA), was employed as a therapeutic agent for drug-drug interactions. According to the present study, nobiletin can ameliorate the renal accumulation of 7−OH MTX through the interaction with aldehyde oxidase. RNA-seq analysis revealed that 7−OH MTX was mainly related to protein processing in endoplasmic reticulum (ER) stress, with the PERK/CHOP pathway selected as the most significant for metabolic nephrotoxicity. Meanwhile, the cross-linked proteins and conducted signals were investigated by western blotting and further verified by GSK inhibition analyses. These results indicated that nobiletin protected renal function from MTX-induced nephrotoxicity by modulating metabolism and ameliorated the metabolic toxicity of 7−OH MTX on ER stress-induced PERK/CHOP conduction by maintaining Ca2+ homeostasis and reducing the production of reactive oxygen species. [ABSTRACT FROM AUTHOR]