AbstractIsatis indigotica Fort. is extremely widely planted and used for the source of the traditional Chinese medicine Radix Isatidis (Ban-Lan-Gen), which had pharmacological activities such as anti-endotoxic, antibacterial, antinociceptive, anti-inflammatory and antipyretic. In the cultivation, salt stress could affect the yields of Radix Isatidis, and activate the secondary metabolism, eg. the contents of epigoitrin in root and indigo in leaf increased after a period of salt stress, which was possibly regulated by related genes. But in early salt stress, the molecular response of I. indigotica and expression of related genes are also worthy of our attention, which it helps us to more fully understand the molecular response mechanism of I. indigotica, so that we can make better use of “salt stress” to stimulate its secondary metabolism. To determine the molecular changes of I. indigotica in salt stress, the RNA-seq was carried out for the expression profile with NGS QC Toolkit (v2.3.2) software, the Illumina HiSeqTM 2000 in Biomarker Technologies Co., Ltd and ESTScan program. The de novo assembly resulted in 33109 unigenes from more than 18.71 Gb data. Of these, 28868 unigenes were annotated using KOG, KEGG, Nr, Nt, Swiss-Prot and TrEMBL databases. Then, 11829 simple sequence repeats were found in unigenes and 7725 primers were designed. After detecting the expression value, the edgeR package found 135 DEG, of those 48 were up-regulated and 87 were down-regulated. The expression pattern of genes that was validated by qPCR indicated that mitochondrial transcription termination factor, non-LTR retroelement reverse transcriptase and CYP79F1 which involved in the first step in biosynthesis of core short-chain aliphatic glucosinolates might play a vital role in coping with salt stress. Although one assembly of I. indigotica unigenes were obtained before, this study would provide more molecular data for further analysis, and facilitate studies on the functions of genes involved in the salt related signal pathways.