Uranium (U) contamination of soil has become a major concern with respect to its toxicity, accumulation in the food chain, and persistence in the environment. Anthropogenic activities like mining and processing of U ores has become pressing issues throughout the world. The aim of the work is to understand the chemical fractionation of U in polluted soil and the mechanism involved. U-free soils samples of eluvial (E), illuvial (B), and parent-material (C) horizons from a hydrometallurgical factory area were used. The experimental results showed that the U adsorption capacity decreased with depth, and its mobility in the upper soil is better than the lower. It was closely related to distribution coefficient (K), pH, organic-matter (OM), and carbonate content of soil horizons. The chemical fractionation of U was studied using the BCR sequential extraction scheme for soils after saturated adsorption. It was noted that the U reducible and oxidizable fraction in the E and B horizons can vertically transfer to the C horizon and occurs a significant rearrangement of U in different horizons. BET, SEM, XRD, and FT-IR analyses showed that different U distribution and migration in soil profile is mainly affected by specific surface area, soil particle size, mineral composition, and active groups. The XPS data further indicated that U (VI) is gradually converted to U (IV) with decreased depth and fixed in deeper soil becoming insoluble and immobile. It is the first step to investigate potential migration and plan U mining and milling area long-term management.