ABSTRACTMangiferin (MGN), a C‐glucosylxanthone abundantly found in mango plants, was studied for its potential to ameliorate methylmercury (MeHg) induced mitochondrial damage in HepG2 (human hepatocarcinoma) cell line. Cell viability experiments performed using 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐ diphenyltetrazolium bromide (MTT) showed protective property of MGN in annulling MeHg‐induced cytotoxicity. Conditioning the cells with optimal dose of MGN (50 µM) lowered MeHg‐induced oxidative stress, calcium influx/efflux, depletion of mitochondrial trans‐membrane potential and prevented mitochondrial fission as observed by decrease in Mitotracker red fluorescence, expression of pDRP1 (serine 616), and DRP1 levels. MGN pre‐treated cells demonstrated elevation in the activities of glutathione (GSH), Glutathione‐S‐transferase (GST), Glutathione peroxidase (GPx), Glutathione reductase (GR), reduced levels of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) and mitochondrial electron transport chain (ETC) enzyme complexes. In addition, the anti‐apoptotic effect of MGN was clearly indicated by the reduction in MeHg‐induced apoptotic cells analyzed by flowcytometric analysis after Annexin V‐FITC/propidium iodide staining. In conclusion, the present work demonstrates the ability of a dietary polyphenol, MGN to ameliorate MeHg‐mediated mitochondrial dysfunction in human hepatic cells in vitro. This hepatoprotective potential may be attributed predominantly to the free radical scavenging/antioxidant property of MGN, by facilitating the balancing of cellular Ca2+ ions, maintenance of redox homeostasis and intracellular antioxidant activities, ultimately preserving the mitochondrial function and cell viability after MeHg intoxication. As MeHg intoxication occurs over a period of time, continuous consumption of such dietary compounds may prove to be very useful in promoting human health. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 630–644, 2017.