To identify a terpene synthase that catalyzes the conversion of geranyl pyrophosphate (GPP) to alpha-pinene and is involved in the biosynthesis of paeoniflorin. Two new terpene synthase genes were isolated from the transcriptome data of Peaonia lactiflora. Phylogenetic analysis and sequence characterization revealed that one gene, named PlPIN, encoded a monoterpene synthase that might be involved in the biosynthesis of paeoniflorin. In vitro enzyme assay showed that, in contrast to most monoterpene synthases, PlPIN encoded an alpha-pinene synthase which converted GPP into alpha-pinene as a single product. This newly identified alpha-pinene synthase could be used for improving paeoniflorin accumulation by metabolic engineering or for producing alpha-pinene via synthetic biology.