In vitro culture parameters were investigated in order to develop an effective protocol for Artemisia annua cultivation and artemisinin production. The explants, shoot tips and axillary buds, were individually cultured on MS medium contained 6-benzylaminopurine (BAP) or growth regulators. Treatment with mercuric chloride (1.0%) for 5 min gave the highest survival percentage (86.0% and 90.0%) and the lowest contamination percentage (40.0% and 64.0%) for terminal shoot tip and axillary bud, respectively. Axillary bud explants surpassed shoot tips in development and direct regeneration. High concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA) plus 0.5 mg/L BAP were suitable for callus induction while low concentrations and the control induced less necrosis and more explant development, as well as increased artemisinin concentration. Lower BAP concentrations induced an increased growth rate, while the higher BAP concentration encouraged proliferation. Seven treatments with different concentrations of NAA, 2,4-D, and salicylic acid produced lower amounts of artemisinin than that produced by control (0.5 mg/L BAP). Decreasing level of artemisinin could be due to reduced rates of plant growth and decreased amounts of green matter by growth regulators, which may affect the plastidic pathway for artemisinin production