Knowledge of the sorption and speciation of uranium at mineral/water interface is essential to predict its long-term behavior. The sorption of uranium on minerals is well known affected by its interaction with EDTA. Here, the sorption of U(VI) on rutile in the presence or absence of EDTA was investigated by combining batch sorption, species calculation, time-resolved fluorescence spectroscopy (TRFS) and X-ray photoelectron spectroscopy (XPS). The batch sorption results suggest that the sorption of U(VI) on rutile is governed by outer-sphere surface complexation under acidic conditions, and undergoes a transformation to inner-sphere surface complexation as the pH increases. In the presence of EDTA, the sorption process is dominated by inner-sphere interactions across the entire pH range. EDTA has been observed to reduce the sorption of U(VI) on minerals, thereby enhancing the mobility of U(VI). Greater inhibition of U(VI) sorption was observed with increasing concentration of EDTA. The TRFS and XPS analysis reveal that the uranium presents as entirely distinct surface complexes on rutile in the presence or absence of EDTA. These findings are essential for understanding the sorption mechanism of U(VI) with EDTA at a molecular scale and developing a reliable assessment for the long-term storage of radioactive waste.