Salvia miltiorrhiza Bunge, a perennial plant of Lamiaceae, accumulates abietane-type diterpenoids of tanshinones in root, which have been used as traditional Chinese medicine to treat neuroasthenic insomnia and cardiovascular diseases. However, to date the biosynthetic pathway of tanshinones is only partially elucidated and the mechanism for their root-specific accumulation remains unknown. To identify enzymes and transcriptional regulators involved in the biosynthesis of tanshinones, we conducted transcriptome profiling of S. miltiorrhiza root and leaf tissues using the 454 GS-FLX pyrosequencing platform, which generated 550,546 and 525,292 reads, respectively. RNA sequencing reads were assembled and clustered into 64,139 unigenes (29,883 isotigs and 34,256 singletons). NCBI non-redundant protein databases (NR) and Swiss-Prot database searches anchored 32,096 unigenes (50%) with functional annotations based on sequence similarities. Further assignments with Gene Ontology (GO) terms and KEGG biochemical pathways identified 168 unigenes referring to the terpenoid backbone biosynthesis (including 144 MEP and MVA pathway genes and 24 terpene synthases). Comparative analysis of the transcriptomes identified 2,863 unigenes that were highly expressed in roots, including those encoding enzymes of early steps of tanshinone biosynthetic pathway, such as copalyl diphosphate synthase (SmCPS), kaurene synthase-like (SmKSL) and CYP76AH1. Other differentially expressed unigenes predicted to be related to tanshinone biosynthesis fall into cytochrome P450 monooxygenases, dehydrogenases and reductases, as well as regulatory factors. In addition, 21 P450 genes were selectively confirmed by real-time PCR. Thus we have generated a large unigene dataset which provides a valuable resource for further investigation of the radix development and biosynthesis of tanshinones.