Background <p><i>Isodon rubescens</i>is used in traditional Chinese medicine and has drawn attention as a rich source of oridonin, a compound reported to possess medicinal properties. Differential transcriptome analysis is known to be an effective method for gene selection of diterpene oridonin biosynthetic pathways, however, the MeJA-induced genetic responses of <i>I. rubescens</i> have not yet been analyzed.</p> Results <p>In this study, comparative transcriptome analysis of JY (with oridonin) and LS (no oridonin) lines of <i>I. rubescens</i> with or without a methyl jasmonate (MeJA) treatment revealed 43,115 assembled unigenes, 11,253 of which were differentially expressed. We also identified a total of 5,070 statistically significant differentially expressed unigenes which respond to MeJA including 397 transcription factor (TF) unigenes, such as bHLH, ERF, NAC, and MYB. Furthermore, assignments with KEGG pathways identified 18 of 53 unigenes referring to the diterpenoid biosynthesis, 10 cytochrome P450 unigenes, and three different expression TF unigenes were speculated that these genes could regulate the synthesis of oridonin based on MeJA induced. Finally, 18 unigenes were validated by qRT-PCR, and their expression levels were matched with the sequence data. In addition, Single Nucleotide Polymorphisms (SNPs) were analyzed and 14,609 simple sequence repeats (SSRs) were detected in <i>I. rubescens</i>, providing further support for future genetic variation and marker-assisted selection.</p> Conclusions <p>This is the first time to study the response of related genes to MeJA treatment in <i>I. rubescens</i>, and some new genes were putatively involved in oridonin biosynthesis in <i>I. rubescens</i>. Our results provides a valuable gene resource to functionally characterize of response to MeJA and oridonin biosynthesis in <i>I. rubescens</i>.</p>