###### Abstract  
_Scutellaria barbata_ (Lamiaceae) is an important medicinal herb widely used in China, Korea, India, and other Asian countries. _Neo_-clerodane diterpenoids are the largest known group of _Scutellaria_ diterpenoids and show promising cytotoxic activity against several cancer cell lines. Here, Illumina-based deep transcriptome analysis of flowers, the aerial parts (leaf and stem), and roots of _S. barbata_ was used to explore terpenoid-related genes. In total, 121,958,564 clean RNA-sequence reads were assembled into 88,980 transcripts, with an average length of 1370 nt and N50 length of 2144 nt, indicating high assembly quality. We identified nearly all known terpenoid-related genes (33 genes) involved in biosynthesis of the terpenoid backbone and 14 terpenine synthase genes which generate skeletons for different terpenoids. Three full length diterpene synthase genes were functionally identified using an in vitro assay. _SbTPS8_ and _SbTPS9_ were identified as normal-CPP and _ent_-CPP synthase, respectively. _SbTPS12_ reacts with _SbTPS8_ to produce mitriadene. Furthermore, _SbTPS12_ was proven to be a less promiscuous class I diterpene synthase. These results give a comprehensive understanding of the terpenoid biosynthesis in _S. barbata_ and provide useful information for enhancing the production of bioactive _neo_-clerodane diterpenoids through genetic engineering. _Scutellaria barbata_; transcriptomics analysis; terpenoid backbone biosynthesis; terpenine synthase; diterpene synthase 2018 23 2952 2312952(Figure 1B) are two typical metabolites isolated from _S. barbata_ that have significant inhibitory effects on lipopolysaccharide-induced nitric oxide production [10] and inhibition against Epstein-Barr virus lytic replication [11]. In addition to the general characteristics of triterpen in Lamiaceae as a whole, they are also especially renowned for essential oils only common in the subfamily Nepetoideae [1]. The volatile oils in this subfamily mainly consist of monoterpenoids and sesquiterpenoids. Iridoids, which are monoterpenoid lactones, are also produced in most species of the Lamioideae, Viticoideae, Ajugoideae, and Scutellarioideae. A recent study investigated the diversity mechanism of the three terpenoid classes (monoterpenes, sesquiterpenenes, and iridoids) in Lamiaceae [12]. It showed that multiple mechanisms contributed to the evolution of chemodiversity. Gene family expansion rather than increased enzyme promiscuity of terpene synthases is correlated with mono- and sesquiterpene diversity and differential expression of core genes within the iridoid biosynthetic pathway is associated with iridoid presence/absence. However, besides the triterpen reported from _S. barbata_, little is known about the monoterpenes and sesquiterpenens in _S. barbata_. Despite their structural diversity, all terpenoids begin with the five-carbon building blocks isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). In plants, both IPP and DMAPP are synthesized by two independent pathways, the mexulonic acid (MVA) and methylerythritol phosphate (MEP) pathways. However, metabolic cross-talk via the exchange of IPP and to a lesser extent DMAPP is observed between these two pathways in both directions [13; 14; 15]. Recently, an alternative MVA pathway was found. These studies showed that isopentenyl phosphate kinase (IPK) can reactivate isopentenyl phosphate (IP) via ATP-dependent phosphorylation, thus forming the primary metabolite IPP and altering conventional views of metabolic regulation of terpenoid metabolism in plants [16; 17; 18]. IPP and DMAPP are subsequently used in multiple compartments by short-chain prenyltransferase (GPP, FPP, and GGPP synthases) to produce prenyl diphosphate intermediates, including geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15), and geranylgeranyl diphosphate (GGPP, C20). These precursors are further synthesized by a family of terpene synthases (TPSs) to form 10-carbon monoterpenes, 15-carbon sesquiterpenes, and 20-carbon diterpenes.