Abstract: Cadmium (Cd) is a common pollutant not required for life activities, which can have extremely strong toxicity to organisms and affect the growth of crops at a low concentration in soil. To investigate the molecular mechanism of soybean root responding to Cd stress, 7-day old soybean seedlings were stressed by Cd (75 μmol·L^-1) for 0 , 4 , 8, 12 and 48 h. Comparative transcriptome analysis showed 244, 1545, 442 and 1401 of genes responded to the four Cd treatments, respectively, and total 2670 differential expression genes were obtained. GO analysis classified these genes into 56 functional categories and COG analysis classified them into 25 functional categories. KEGG analysis showed that many genes involved in the phenylalanine metabolism, ubiquinone and other terpenoid-quinone biosynthesis and cysteine and methionine metabolism and so on. Further we found that expression of three isoflavones 2′-hydroxylase genes, two isoflavonereductase genes and a chalcone synthase gene were evidently up-regulated in all Cd treatments. The results of RT-PCR analysis of four DEGs were consistent with those of RNA-Seq data, further confirming the reliability of RNA-Seq results.