Good boron removal and isotopic separation behavior was observed on magnetic magnetite nanoparticles (MMNs) in a previous study, but the mechanism of boron adsorption and isotopic separation remains unclear. Here, experimental studies accompanied by quantum chemistry calculations were implemented for the first time to reveal the nature of boron adsorption and isotopic separation on MMN. First, the fitted peaks of Fe2p of MMN by X-ray photoelectron spectrometry (XPS) before and after adsorption showed decrease in Fe(II)/Fe(III) ratio, which validated the formation of Fe-O-B interaction. Fe-O-B bond was also observed through fitted peaks of O1s and dihydroxy complexation where MMNs prefer to adsorb [B(OH)(4)](-)(H3BO3) at pH < = pH(zpc) (pH > pH(zpc)) identified by the ratio of integrated peaks. ATR-FTIR revealed the hydroxyl moiety as the main adsorptive group. The selectivity of MMN toward [B(OH)(4)](-) at pH < = pH(zpc) and toward H3BO3 at pH > pH(zpc) was figured out by calculating the ratio between the integrated areas of B-O bands at different pH values. As a result, the trend of adsorption capacity as well as the isotopic separation factor with pH was well illustrated. The concluded adsorption mechanism from the experiment was further verified with the simulated adsorption energies (Delta E) and isotopic separation factors (S) calculated by DFT quantum simulation. [GRAPHICS] .