<p>Soils developed on carbonates are susceptible to geogenic enrichment of potentially toxic trace metals (TMs) and thus present potential risks to the ecosystem and population. Clarifying factors that influence the bioavailability of TMs is crucial for soil risk control. This study aimed to investigate the bioavailability and speciation of TMs in limestone-derived soils in southwestern China where a large area was recognized as high geochemical background. The total, bioavailable content and speciation of TMs were analyzed by acid digestion, single and sequential extraction, respectively. The results showed that high contents of TMs in the acid-insoluble limestone residues resulted in the geogenic enrichment of Cr, Ni, Pb and Zn in soils. The risk assessment results showed that the ecological risks of TMs in the studied soils were low, except Cd. The DTPA extraction showed that the bioavailability of Pb and Cu was higher than Cr, Ni, and Zn, and the bioavailability of Pb and Cu in paddy soils was twice as high as in upland soils. In paddy soils, Cr, Ni and Zn reside in residual and Fe-oxides bound fractions, Cu and Pb are primarily hosted by residual, organically and Fe–Mn oxides bound fractions. Additionally, the mobility and bioavailability of Cu and Pb increased towards the surface. Soil organic matter is identified as a critical factor influencing the bioavailability of Pb and Cu. These findings highlight the potential risks of mobilizing geogenic TMs induced by organic matter in paddy soils.</p>