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Lactoferrin is a minor whey protein known for its multifunctionalities, particularly for its rule in regulation of immune system and for its antimicrobial activities. Although bovine lactoferrin has been extensively studied, very few information exists on lactoferrin from mare's milk. This article studies the antioxidant properties via metal chelation of purified lactoferrin from Kazakhstan mare's milk. Equine lactoferrin shows free radical-scavenger activity when tested with radical DPPH and ABTS and has reducing capacities testing by FRAP. Moreover, the ferrous(II) and copper(II) ion-chelating power is highlighted by chemical colorimetric tests. Finally, the newly introduced biophysics switchSENSE® technology allowed to measure in real time the molecular interactions between equine lactoferrin and divalent cations. The order of magnitude of the K D values was tens or so μM for the four metal ions tested: Zn2+ (K D = 23.9 ± 4.0 μM), Ca2+ (K D = 28.2 ± 4.4 μM), Cu2+ (K D = 43.5 ± 5.1 μM) and Fe2+ (K D = 54.1 ± 8.9 μM). According to these results, we can conclude that equine lactoferrin shows antioxidant activity by radical-scavenging mechanism, reducing capacities and divalent pro-oxidant metals chelation ability. • Purification of equine lactoferrin, which we purified from Kazakh mare's milk. • Antioxidant activity of equine lactoferrin. • Using complementary approach: radical-scavenging, metal reducing and divalent pro-oxidant metal chelation activities. • Using new biophysics switchSENSE® technology to measure in real time the molecular interactions between two partners. • Determination of association, dissociation and equilibrium constants of the complex metal-lactoferrin. [ABSTRACT FROM AUTHOR]