Tropane alkaloids (TA) including hyoscyamine, anisodamine, scopolamine and anisodine, are used medicinally as anticholinergic agents with increasing market demand, so it is very important to improve TA production by metabolic engineering strategy. Here, we report the simultaneous introduction of genes encoding the branch-controlling enzyme tropinone reductase I (TRI, EU424321) and the downstream rate-limiting enzyme hyoscyamine-6β-hydroxylase (H6H, EF187826) involved in TA biosynthesis into Anisodus acutangulus hairy roots by Agrobacterium-mediated gene transfer technology. Transgenic hairy root lines expressing both TRI and H6H (TH lines) produced significantly higher (P < 0.05) levels of TA compared with the control and single gene transformed lines (T or H lines). The best double gene transformed line (TH53) produced 4.293 mg g⁻¹ TA, which was about 4.49-fold higher than that of the control lines (0.96 mg g⁻¹). As far as it is known, this is the first report on simultaneous introduction of TRI and H6H genes into TA-producing plant by biotechnological approaches. Besides, the content of anisodine was also greatly improved in A. acutangulus by over-expression of AaTRI and AaH6H genes. The average content of anisodine in TH lines was 0.984 mg g⁻¹ dw, about 18.57-fold of BC lines (0.053 mg g⁻¹ dw). This is the first time that this phenomenon has been found in TA-producing plants.