Mangiferin is a natural xanthonoid with various biological activities. Quantification of mangiferin in fruit peel, pulp, and seed kernel was carried out in 11 Chinese mango (Mangifera indica L.) cultivars. The highest mangiferin content was found in the peel of Lvpimang (LPM) fruit (7.49 mg/g DW). Efficient purification of mangiferin from mango fruit peel was then established for the first time by combination of macroporous HPD100 resin chromatography with optimized high-speed counter-current chromatography (HSCCC). Purified mangiferin was identified by both HPLC and LC-MS, and it showed higher DPPH center dot free-radical scavenging capacities and ferric reducing ability of plasma (FRAP) than by L-ascorbic acid (Vc) or Trolox. In addition, it showed significant protective effects on human umbilical vein endothelial cells (HUVEC) under H2O2-induced stress. Cells treated with mangiferin resulted in significant enhanced cell survival under of H2O2 stress. Therefore, mangiferin from mango fruit provides a promising perspective for the prevention of oxidative stress-associated diseases.