Isomorphism is a common phenomenon in clay minerals, which is one of the main reasons for the change of mineral chemical composition and physical and chemical properties. In this study, four kinds of palygorskite with different degrees of isomorphic replacement of octahedral cations were selected. The microstructure and surface-interface properties of different Mg2+/Al3+ palygorskite were analyzed by X-ray diffraction, infrared spectroscopy, N2 adsorption-desorption method, X-ray fluorescence spectroscopy and other characterization methods, and the effects of isomorphic replacement on microstructure such as Mg content and surface-interface properties such as hydroxyl concentration and acid content were discussed. Taking Cu2+ as the target pollutant, the adsorption capacity of different palygorskite to Cu2+ was measured and discussed. Combining the surface hydroxyl concentration and acid amount of palygorskite, as well as the concentration, time and temperature of the reaction system, the rule of adsorption of the four palygorskite for Cu2+ was Mg-rich palygorskite (a-ATP) > palygorskite with equal Mg and Al (c-ATP) > Al-rich palygorskite (d-ATP) > palygorskite with the most Mg (b-ATP), demonstrating that when Mg was partially replaced by Al, palygorskite contained appropriate hydroxyl concentration and acid amount, resulting in the best adsorption effect for Cu2+ (29.2 mg/g). It is of great significance for resource utilization of palygorskite to reveal the regularity of heavy metal adsorption by isomorphic replacement of palygorskite.