Abstract Antimony (Sb) contamination of soils and its potential negative impact on crop yields have been recently recognized. To explore the effects of Sb(III) on wheat, responses of physical growth properties, enzyme activities and gene expression were examined. The root length was the most relevant indicator to exposure concentration, the root fresh weight was the most sensitive endpoint according to the comparison of the EC 50 (50% inhibitory effect concentration) values, and NRAMP1 gene expression at 1 mg L−1 was more sensitive than other genes. The genes HMA2 and IRT1 were upregulated but less sensitive than NRAMP1. Additionally, the antioxidant enzyme response was examined to explore Sb toxicity toward wheat. The glutathione (GSH) content in roots exposed to high concentrations of Sb(III) was higher than that in the control group, which indicated that GSH is involved in protecting wheat against the effects of Sb(III). Comparing the different antioxidant responses and correlations with malondialdehyde (MDA) in different tissues, super oxide dismutase (SOD) exhibited the main contribution to defense Sb(III) in roots; SOD and peroxidase (POD) were the major antioxidants in shoots. Gene expression of transporter proteins was a highly sensitive indicator of Sb toxicity in wheat and allowed for understanding the defense mechanism in wheat. The upregulation of HMA2 under Sb(III) expression was one kind of defense mechanism, which can induce efflux of heavy metals into the apoplast or vacuoles. Biomass and plant height results suggested that wheat may grow better than other crops under similar exposure due to the lower degree of Sb uptake. This work is the first to provide a wheat uptake model of Sb(III) under a wide concentration range as well as a perspective on the response to this stress at the molecular level. Graphical abstract Unlabelled Image Highlights • Root length was a good indicator for estimating the Sb(III) exposure concentration. • The different Sb (III) uptake models of wheat roots and shoots were investigated. • The wheat antioxidant system was activated by Sb(III) and the main antioxidants in shoots and roots were different. • Lower level Sb(III) promoted the wheat root growth, but it regulated the main metal(loid) transporter genes expression. [ABSTRACT FROM AUTHOR]