Pueraria lobata (willd.) Ohwi is a consumable selenium-enriched plant used for medicinal purposes. The molecular response to selenium (Se) stimuli in P. lobata is currently unknown. We used RNA-Seq to identify potential genes involved in selenite metabolism and analyzed their expression profiles. We obtained a total of 150,567 unigenes, of which 90,961 were annotated, including 16 structural genes, 14 sulfate transporters, and 13 phosphate transporters that may be involved in Se metabolism, and 33 candidate structural genes involved in isoflavone biosynthesis. The genes with a —foldchange— >2 and q value <0.05 after sodium selenite treatment were identified as differentially expressed genes (DEGs). We obtained a total of 4,246 DEGs, which were enriched in GO terms that included “response to stimulus”, “response to stress”, “signal transduction”, “response to abiotic stimulus”, and “response to chemical”. Of the 4,246 DEGs, one sulfate transporter and five phosphate transporter genes involved Se metabolism, and nine structural genes involved in isoflavone biosynthesis were up-regulated. The expression patterns of 10 DEGs were selected randomly and validated using qRT-PCR. The Pearson Correlation Coefficient (r) was 0.86, indicating the reliability of RNA-Seq results. 22 Reactive Oxygen Species (ROS) scavenging DEGs were found, 11 of which were up-regulated. 436, 624 transcription factors (TFs) correlated with structural genes were identified that may be involved in Se and isoflavone biosynthesis, respectively, using r ( r > 0.7 or r < − 0.7). 556 TFs were related to at least one sulfate and phosphate transporter. Our results provided a comprehensive description of gene expression and regulation in response to Se stimuli in P. lobata .