This study explored the mechanism of antimony (Sb) isotopic fractionation during chemical weathering and soil biogeochemical processes, which have important implications for Sb isotopic composition in the hydrosphere. We analyzed the major and trace element contents and Sb isotopic composition along an 8 m deep basalt weathering profile in the Huguangyan area of Guangdong Province, southern China. Mineral adsorption experiments were conducted to determine the mechanism of Sb isotopic fractionation in the weathering profile. The following minerals, α-FeOOH, γ-Fe₂O₃, β-MnO₂, γ-Al₂O₃, montmorillonite-K10, and kaolinite-1A, preferentially adsorbed light Sb isotopes, and increased pH may lead to a lighter isotopic composition of adsorbed Sb. The positive values of τSbTiO2 in the upper profile reflect exogenous Sb inputs, such as rainfall and litterfall. Accordingly, Sb isotopic fractionation is controlled by Sb adsorption onto organic matter and Fe (hydr)oxides in surface soil. At a depth of 1.8 m, Sb and other redox-sensitive elements, such as Fe, Mn, Ce, Co, Cr, and Mo, were distinctly enriched. Changes in the redox conditions result in strong Sb isotopic fractionation. In the middle layer (2–4 m), the combined effects of changes in organic matter content, soil pH, and weathering intensity explains the enrichment of light Sb isotopes. The bottom layer of the profile did not receive exogenous Sb and exhibited an Sb isotopic fractionation pattern similar to that of the basaltic bedrock. In the bottom layer, secondary Fe (hydr)oxides (e.g., goethite and hematite) preferentially adsorb the light Sb isotope (¹²¹Sb), whereas the heavy Sb isotope (¹²³Sb) is released into the aquatic environment during intensive chemical weathering.