Abstract: Atractylodes macrocephala is an important medicinal herb belonging to the family compositae. In order to provide efficient pathway for commercial production of Atractylodes macrocephala, obtain medicinal secondary metabolite to meet great demand in herbal medicine, and avoid environment destruction induced by tradition cultivation mode of destroying native vegetation, a method for rapid micropropagation of Atractylodes macrocephala through plant regeneration by leaf and direct shoot multiplication were established in the paper .The effect of different cytokinin and concentration on callus introduction from leaf and direct shoot multiplication and the influence of different light and temperature on subcultured callus browning were investigated. The results showed that the highest rate of direct shoot multiplication (4.55 per bud ) was formed at MS+ 6-BA1 mg/L +NAA0.2 mg/L. The optimal substrate for callus introduction and plant regeneration from leaf was MS +KT 1mg/L+NAA0.2 mg/L where the highest callus introduction (96.7%) and proliferation (90%) were obtained. Different cytokinin and variable concentration had significant differences. The effect of KT on callus induction and proliferation was superior to 6-BA, whereas 6-BA was more efficient than KT at higher concentration of 2mg/L on direct shoot multiplication. Callus browning was related to different treatments of temperature and light, and it was availably controlled when cultured under 1000 lx light intensity and 20±0.5℃ temperature.