Atropa belladonna L. is the commercial plant material for production of tropane alkaloids, including hyoscyamine and scopolamine. The wild-type Atropa belladonna is characterized by the hyoscyamine-rich chemotype, in which the hyoscyamine content is much higher than the scopolamine content. It is the common goal for the pharmaceutical industry to increase the content of scopolamine in A. belladonna. Based on the T0 progeny of transgenic A. belladonna with NtPMT and HnH6H overexpression, T1 progeny of transgenic A. belladonna were obtained through self-pollination and used in a field trial. The 461 bp fragment of NtPMT and the 1 077 bpHnH6 H were simultaneously expressed from T1 progeny of transgenic A. belladonna, but were not obtained from the wild-type A. belladonna. At the transcription level, the expression of NtPMT and HnH6H were detected in T1 progeny of transgenic A. belladonna, but were not detected in the wild-type plants. Further, the alkaloids were analyzed by HPLC. In the stems and leaves of T1 progeny of transgenic A. belladonna, hyoscyamine was not detected and scopolamine was detected at very high levels; in the stems and leaves of wild-type A. belladonna, hyoscyamine was detected at much higher levels. In the leaves of T1 progeny of transgenic A. belladonna, the content of scopolamine was 15-36 folds higher than that of wild- type leaves; in the stems of T1 progeny of transgenic A. belladonna, the scopolamine content was 37-108 folds higher than that of wild-type stems. In conclusion, overexpression of NtPMT and HnH6H greatly enhanced conversion of hyoscyamine into high-value scopolamine and improved the commercial value of A. belladonna.