Various nutrients (Mg, Zn, Fe, Mn, Si, etc.) can supress cadmium (Cd) uptake and alleviate Cd toxicity, but the mechanisms are not the same. In this study, the molecular mechanism governing the effects of boron (B) on uptake of Cd in hydroponically grown wheat was characterized. As compared to control (0 μM Cd), B concentration per plant decreased by 22% and 29% under 5 μM Cd and 50 μM Cd treatment respectively. In addition, B application decreased Cd concentration and accumulation in whole wheat. Correlation analysis of different elements show that there was a highly negative correlation between concentrations of B and Cd (r = −0.854 with significant correlation) in wheat. Additionally, 16,543 differentially expressed genes (DEGs) (7666 up- and 8877 down-regulated) were detected between 0 and 5 μM Cd treatments in wheat roots by transcriptome sequencing. Gene ontology functional category and Kyoto encyclopedia of genes and genomes pathway analyses indicated that the DEGs were involved in biological process, cellular component, and molecular function. Five highly homologous genes to Cd transporters were identified; these genes were involved in metal ion binding, transmembrane ion transport, and protein transport. According to the qRT-PCR results, expression of all these genes was down-regulated in the 462 μM of B treatment compared with the 46.2 μM of B treatment regardless of the Cd treatments (0.5 or 5 μM Cd). These results suggest that B is an inhibitor of Cd uptake, and the down-regulation of five highly homologous genes could be associated with decreased uptake of Cd after B application.