Abstract: Tropinone reductase I (TRI) is a key branch point enzyme in the midstream of tropane alkaloids (TAs) biosynthesis pathway and represents an important target for TAs metabolic engineering, which can lead to metabolic flux of substrate tropinone to TAs. A novel TRI gene was isolated from Datura arborea, a woody resource plant, and designated as DaTRI2 (GenBank accession number is MH705164). The full-length cDNA of DaTRI2 with 1 135 bp exhibits a high sequence homology (96.8%) with DaTRI, and is predicted to encode a protein of 347 amino acids. Deduced DaTRI2 protein contain a conserved TGXXXGXG motif involved in NADPH binding, the catalytic N-S-Y-K tetrad motif and eleven amino acid residues important for binding to its substrate tropinone. The phylogenetic analysis revealed that DaTRI2 and other TRIs from Solanaceous plants belong to the same cluster and DaTRI2 exhibited closest phylogenetic proximity to TRIs from Datura. DaTRI2 was expressed in E. coli and the purified recombinant protein can catalyze both tropinone reduction and tropine oxidation with an optimum pH value of 8.0 and 9.6, respectively. When tropinone was used as the substrate, the K_m and V_max values of DaTRI2 at pH 6.4 were 210.05 μmol·L^-1 and 69.6 nkat·mg^-1 protein respectively, while the K_m and V_max values for tropine as the substrate were 188.03 μmol·L^-1 and 114 nkat·mg^-1 protein respectively, at pH 9.6. DaTRI2 transcript was most abundant in the young leaf, followed by the root. Cloning of DaTRI2 gene and biochemical analysis of recombinant DaTRI2 facilitate further research on the molecular mechanism on TAs biosynthesis in woody plants and provide a more potent candidate for TAs metabolic engineering.