Abstract Antimony (Sb), a non-essential heavy metal, exerts severe toxic effects on the growth and development of plants. This study investigated the response of Brassica napus to Sb(III) stress under hydroponic conditions, focusing on Sb accumulation, physiological indexes, and transcriptome sequencing. Sb accumulation in different B. napus varieties showed consistent trends with physiological indicators (SOD, POD, CAT, MDA) in XZY512 root tissue. Both parameters increased with Sb concentration, reaching a peak at 75 mg/L before declining, suggesting that 75 mg/L Sb may be the optimal concentration for B. napus adaptation. Transcriptomic analysis identified 8,802 genes in root tissues and 13,612 genes in leaf tissues responsive to Sb stress, predominantly involved in oxidative stress responses, ABC transporters, glutathione metabolism, plant hormone signaling, and MAPK pathways. Physiological index changes were associated with upregulation of genes linked to antioxidants, including as CATs, GPXs, PERs, and GSTUs, in root tissues, whereas photosynthesis-related genes were mostly downregulated in leaf tissues. This work shows the potential of B. napus for phytoremediation efforts and offers important insights into its response mechanisms to Sb stress.