Artemisia sieberi has an important pharmaceutical compound artemisinin. Artemisinin is one of the most natural and strongest antimalarial agent. In this study, for in vitro plant regeneration, leaves from one month-old sterile plants as explants were used in plant media containing different combination hormones, including 2, 4-D, IAA, NAA, BA, and TDZ. The highest indirect regeneration frequency was obtained using 2 mg/l BA, 0.05 mg/l NAA and 2 mg/l BA, 0.5 mg/l IAA. The highest direct regeneration was observed in 0.1 mg/l TDZ. Also, an efficient transgenic root induction system for Artemisia sieberi was developed through evaluating some factors such as Agrobacterium rhizogenes strain, explant type, pre-culture period (0, 24 and 48 hours), co-cultivation period (24, 48 and 72 hours), absence or presence of acetosyringone on the induction rate of transgenic roots were evaluated. The highest roots induction was obtained respectively in MSU440 (83.33%) and ATCC15834 (73%) onto the 4-week-old leaf explants followed by a co-cultivation period of 2 days on a ½ MS medium containing 100 μM acetosyringone. In order to molecular validation of transgenic root, PCR with specific primers of rolB gene was performed. Approximately 0.048 % artemisinin was detected by HPLC analysis in root cultures. Transgenic roots can consider as a new alternative approach of artemisinin production.