To separate siderite from carbonate-bearing iron ore efficiently, specific adsorption configurations and effective species were investigated to compare the adsorption differences of sodium oleate (NaOL) on siderite and hematite surfaces. The species were analysed in neutral conditions, under which the direct flotation of siderite from hematite and quartz was carried out. Changes in the zeta potential, Fourier-transform infrared spectroscopy spectra and X-ray photoelectron spectroscopy spectra caused by the adsorption of oleate species were compared to confirm the adsorbed species on siderite and hematite. Molecular dynamics simulations were carried out to further verify the specific adsorption configurations. Chemisorption of oleate species was detected on both siderite and hematite. The differences in the relative concentration changes of the elements indicate that NaOL worked on siderite and hematite via adsorption of various groups or species. The main functional species were RCOO- for siderite and RCOOH-RCOO- for hematite. A strong chemical bond formed as RCOO- directly acted upon the Fe of siderite. The carbon chains bent and interweaved to form the hydrophobic layer on siderite. The two chains of RCOOH-RCOO- laid upon hematite via weak chemical bonds and hydrogen bonds. Van der Waals and hydrophobic forces were the main interforces of the two chains.