The appropriate timing of bolting and flowering is one of the keys to the reproductive success of Isatis indigotica. Several flowering regulatory pathways have been reported in plant species, but we know little about flowering regulatory in I. indigotica. In the present study, we performed RNA-seq and annotated I. indigotica transcriptome using RNA from five tissues (leaves, roots, flowers, fruit, and stems). Illumina sequencing generated 149,907,857 high-quality clean reads and 124,508 unigenes were assembled from the sequenced reads. Of these unigenes, 88,064 were functionally annotated by BLAST searches against the public protein databases. Functional classification and annotation assigned 55,991 and 23,072 unigenes to 52 gene ontology (GO) terms and 25 clusters of orthologous group (COG) categories, respectively. A total of 19,927 unigenes were assigned to 124 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 80 candidate genes related to plant circadian rhythm were identified. We also identified a number of differentially expressed genes (DEG) and 91 potential bolting and flowering-related genes from the RNA-seq data. This study is the first to identify bolting and flowering-related genes based on transcriptome sequencing and assembly in I. indigotica. The results provide foundations for the exploration of flowering pathways in I. indigotica and investigations of the molecular mechanisms of bolting and flowering in Brassicaceae plants.