AbstractSalvia miltiorrhizais well known for its clinical practice in treating heart and cardiovascular diseases. Its roots, used for traditional Chinese medicine materials, are usually brick-red due to accumulation of red pigments, such as tanshinone IIA and tanshinone I. Here we report aS. miltiorrhizaline (shh) with orange roots. Compared with the red roots of normalS. miltiorrhizaplants, the contents of tanshinones with a single bond at C-15,16 were increased, whereas those with a double bond at C-15,16 were significantly decreased in shh. We assembled a high-quality chromosome-level genome of shh. Phylogenomic analysis showed that the relationship between twoS. miltiorrhizalines with red roots was closer than the relationship with shh. It indicates that shh could not be the mutant of an extantS. miltiorrhizaline with red roots. Comparative genomic and transcriptomic analyses showed that a 1.0 kb DNA fragment was deleted in shhSm2OGD3m. Complementation assay showed that overexpression of intactSm2OGD3in shh hairy roots recovered furan D-ring tanshinone accumulation. Consistently,in vitroprotein assay showed that Sm2OGD3 catalyzed the conversion of cyptotanshinone, 15,16-dihydrotanshinone I and 1,2,15,16-tetrahydrotanshinone I into tanshinone IIA, tanshinone I and 1,2-dihydrotanshinone I, respectively. Thus, Sm2OGD3 functions as tanshinone 15,16-dehydrogenase and is a key enzyme in tanshinone biosynthesis. The results provide novel insights into the metabolic network of medicinally important tanshinone compounds.