Heavy metals (HMs) are abundant in the karst soils of Southwest China, posing significant health risks to millions of people. Iron (Fe) (hyr)oxides serve as critical carriers of HMs in these soils; however, the processes governing Fe oxide formation and transformation associated with HM accumulation during carbonate weathering in karst region is less understood. In this study, we present Fe isotope compositions from a carbonate–derived profile to investigate the major factors controlling Fe migration. In the saprolite layer, strong correlations between δ⁵⁶Fe_bulk and the proportions of extracted Fe_NH2OH.HCl or Fe_residue fractions suggest that the formation of goethite and phyllosilicate may be responsible for variations in δ⁵⁶Fe_bulk. The positive correlations between δ⁵⁶Fe_NH2OH.HCl values and HM concentrations in this layer suggest an enhanced capacity for HM fixation by goethite in these soils. In contrast, the fractionation of Fe isotopes in the soil layer appears to be influenced by vegetation, as indicated by the correlation between total organic carbon and δ⁵⁶Fe_bulk. The negative correlations between δ⁵⁶Fe_bulk values and HM concentrations in the soil layer likely indicate that vegetation litter enhances the retention capacity of Fe oxides for HMs. This study highlights the trajectory of Fe and its connection to HM accumulation in karst soil with high geological background levels.