Antimony (Sb) contamination has attracted global attention due to the elevated Sb entering the environment and threatening human health. Dehydrogenase activity (DHA) has been used as a sensitive bioindicator to assess Sb toxicity. However, only total Sb content and a few types of soils were used so far, which ignored the effect of Sb adsorption characteristics and soil heterogeneity in the Sb toxicity assessment. This study used 18 soil samples varying in their properties to explore the Sb adsorption behavior and the subsequent effects on Sb toxicity using DHA as an indicator. The adsorption isotherm parameters of Sb in 18 soils showed that acidic and neutral soils favored Sb adsorption (n a > 2) over alkaline soils (1 < n a < 2), and the adsorption process tended to be irreversible (R L close to 0). The solid binding force (k L ) of Sb to alkaline soils is less than that of acidic and neutral soils; therefore, alkaline soils pose a higher risk of secondary pollution. Variation partitioning analysis showed that water-soluble Sb was the predominant factor responsible for the observed decrease in DHA. Ecological doses (ED 10 ) based on water-soluble Sb fluctuated from 3.4 to 360 mg kg −1 in soils. Soil properties, such as pH, total phosphorus, amorphous Fe, and clay content, played a crucial role in Sb toxicity to DHA. This study highlighted the importance of water-soluble Sb while evaluating the Sb toxicity in soils.Thus, the study is useful in risk assessment and remediation of Sb-contaminated soils.