Abstract: Based on the genome and transcriptome data of Salvia miltiorrhiza, a cytochrome P450 gene annotated as SmCYP72A395, which was highly expressed in the periderm of S. miltiorrhiza root was cloned. The total length of the cDNA sequence of SmCYP72A395 was 1 578 base pairs, encoding 525 amino acids. The predicted molecular weight of the protein was 59.9 kDa. The physicochemical properties, subcellular localization, protein structure, and conserved domains were predicted by online bioinformatic tools. The theoretical isoelectric point of SmCYP72A395 was 8.68, with a transmembrane domain in the protein. This gene was highly expressed in the flower, leaf, and root periderm tissues of S. miltiorrhiza. To further identify the biological function of SmCYP72A395, the transgenic hairy roots with SmCYP72A395 overexpressed (SmCYP72A395-OE) and RNA interference (SmCYP72A395-RNAi) were constructed, and the content of tanshinone compounds was detected by UPLC between these transgenic lines and control line (haboring the vector plasmid in the transgenic line). Compared with the control line of transgenic hairy roots, the content of dihydrotanshinone Ⅰ, cryptotanshinone, tanshinone Ⅰ and tanshinone ⅡA in the SmCYP72A395-OE lines was significantly less than those in the control line. On the contrary, the content of dihydrotanshinone Ⅰ, cryptotanshinone, and tanshinone Ⅰ was higher in the SmCYP72A395-RNAi lines than those in the control line. These results demonstrated that SmCYP72A395 played a negative role in the regulation of tanshinone accumulation in S. miltiorrhiza. This study lays the foundation for further elucidating the biosynthesis and regulatory pathways of tanshinone compounds in S. miltiorrhiza.