Birnessite is one of the most common manganese oxides in the clay-sized fraction (<2 μm) of soils and has high cation exchange capacity and larger surface area. Birnessite was previously studied for decomposition of selected antibiotics from water. In this study, the removal of tetracycline by birnessite from aqueous solution was investigated as a function of initial tetracycline concentration, solution pH, and temperature, and equilibrium time. Changes in solid phase after tetracycline adsorption and desorption were characterized by X-ray photoelectron spectroscopy, X-ray diffraction, and Fourier transform infrared analyses. Desorption of exchangeable cations accompanying tetracycline removal and partial desorption of tetracycline from birnessite by AlCl3 confirmed that cation exchange was responsible for tetracycline removal at low initial concentrations. Both the external and internal surface areas were readily available for tetracycline uptake by birnessite. The intercalated tetracycline formed a horizontal monolayer configuration in the interlayer of birnessite as deduced from X-ray diffraction analyses.