Mineral-humic complexes are commonly distributed in natural environments and play very important roles in regulating the transport and retention of hydrophobic organic contaminants in soils and sediments. This study examined the structural and conformational changes of humic acid (HA) and mineral-HA complexes after sequential adsorption by minerals, kaolinite, montmorillonite, goethite, and two humins, using UV-Visible spectroscopy, high pressure size exclusion chromatography (HPSEC), Fourier transform infrared (FT-IR) spectroscopy, solid-state ¹³C nuclear magnetic resonance (NMR) spectroscopy, dynamic light scattering, and atomic force microscopy. NMR spectra provided direct evidence for HA fractionation during adsorption onto mineral surfaces, that is, aliphatic fractions were preferentially adsorbed by clay minerals while aromatic fractions were left in solution. We observed a decrease in E₄/E₆ ratio, UV absorbance ratio at 465 and 665 nm, with increasing number of coatings by all adsorbents used in this study, consistent with the NMR results. According to IR and HPSEC results, lignin-like fractions prefer to adsorb onto kaolinite, while polysaccahride fractions are likely to adsorb onto humin. Overall, the fractionation and conformational changes of HA are highly dependent on the surface properties of minerals. Hence, these changes of adsorbed HA may affect the sorption behavior of contaminants.