Polyphenols from Hibiscus sabdariffa (HS) alleviate obesity-related metabolic complications but the metabolites responsible for such effects are unknown. We aimed to elucidate which of the potential plasma metabolites from a polyphenol-enriched HS (PEHS) extract contributed for the reversion of glucolipotoxicity-induced metabolic stress using 3T3-L1 adipocyte and INS 832/13 pancreatic β-cell models under glucolipotoxic conditions. PEHS extract, quercetin (Q) and quercetin-3-O-glucuronide (Q3GA) showed stronger capacity to decrease glucolipotoxicity-induced ROS generation than ascorbic acid or chlorogenic acid. PEHS extract, Q and Q3GA decreased secretion of cytokines (leptin, TNF-α, IGF-1, IL-6, VEGF, IL-1α, IL-1β and CCL2) and reduced CCL2 expression at transcriptional level. In addition, PEHS extract, Q and Q3GA reduced triglyceride accumulation, which occurred through fatty acid synthase (FASN) downregulation, AMPK activation and mitochondrial mass and biogenesis restoration via PPARα upregulation. Electron microscopy confirmed that PEHS extract and Q3GA decreased mitochondrial remodeling and mitophagy. Virtual screening leads us to postulate that Q and Q3GA might act as agonists of these protein targets at specific sites. These data suggest that Q and Q3GA may be the main responsible compounds for the capacity of PEHS extract to revert glucolipotoxicity-induced metabolic stress through AMPK-mediated decrease in fat storage and increase in fatty acid oxidation, though other compounds of the extract may contribute to this capacity. Display omitted •Polyphenol-enriched H. sabdariffa (PEHS) extract alleviates obesity-linked metabolic alterations.•Quercetin forms (Q and Q3GA) are the main flavonols from PEHS extract found in rat plasma.•PEHS extract, Q and Q3GA reduced inflammation and oxidative stress in hypertrophic adipocytes.•PEHS extract, Q and Q3GA reduced fat storage through AMPK-mediated metabolic pathways.•PEHS extract and Q3GA showed capacity to restore mitochondrial viability.