Uranium contamination is of great concern due to its long-term radioactivity and toxicity. The migration of uranium in environment is well known affected by its interaction with clay minerals and the presence of organic matters. The interaction of U(VI) with calcite in the presence of EDTA across weak to strong alkaline conditions was studied by batch sorption and coprecipitation experiments. Multiple spectroscopic methods and DFT calculation were applied to help analyze the experimental results, and the different roles EDTA plays in the interaction process between uranium and calcite were revealed. It is proposed that at hyperalkaline conditions (pH > 9.5), U(VI) favors to precipitate on calcite surface by forming uranium minerals, yet the presence of EDTA impedes the precipitation of uranium by increase the dissolution of calcite by complexing with Ca²⁺. In contrast, under weak alkaline conditions (pH 7.5–9.5), EDTA enhances the removal of uranium from solution by calcite, mainly through increasing the specific surface area and sorption sites of calcite due to the steps, vacancies and/or etch pits created by EDTA on calcite surface without modifying its lattice structure and surface functional groups. DFT calculations of Ca−UO₂ −CO₃ ternary complex interacted with calcite also support the theory that uranyl complexes prefer the sorption on the steps, vacancies and/or etch pits created by EDTA on calcite surface rather than on a flat calcite surface. These results unveil the details of interaction between uranium and calcite, including the presence of EDTA, will help to better understand the migration behavior of uranium in nature, especially in soil minerals.