Salvia miltiorrhiza is one of the most widely used traditional medicines. Natural antisense transcripts (NATs) are a class of long noncoding RNAs that can regulate gene expression. Here, we identified 812 NATs, including 168 cis-NATs and 644 trans-NATs from twelve root, flower, and leaf samples of S. miltiorrhiza using RNA-seq. The expression profiles for 41 of 50 NATs and their sense transcripts (STs) obtained from RNA-Seq were validated using qRT-PCR. The expression profiles of 17 NATs positively correlated with their STs. GO and KEGG pathway analyses mapped the STs for cis-NATs to pathways for biosynthesis of secondary metabolites. We characterized four NATs in detail, including NAT0001, NAT0002, NAT0004, and NAT00023. Their STs are kaurene synthase-like 1 and the homologs of UDP-glucose flavonoid 3-O-glucosyltransferase 6, UDP-glycosyltransferase 90A1, and beta-glucosidase 40, respectively. The first gene is involved in the biosynthesis of bioactive tanshinones, the next two are involved in anthocyanin biosynthesis, whereas the last is involved in phenylpropanoid biosynthesis. Besides, we found seven STs that are potential targets of miRNAs. And we found two miRNAs including miR156a and miR7208, might originate from NATs, NAT0112 and NAT0086. The results suggest that S. miltiorrhiza NATs might interact with STs, produce miRNAs, and be regulated by miRNAs. They potentially play significant regulatory roles in the biosynthesis of bioactive compounds.