Podophyllum hexandrum Royle is an important medicinal plant that produces podophyllotoxin with anti-cancer properties. In China, it is used as a traditional Chinese medicine. Recent years, unplanned exploiting and collection have led to the disappearance of this species in China and India. Effective methods such as tissue culture should be adopted to conserve it to abtain a large scale. It was an crucial process for the final success of tissue culture that seedlings with roots and true leaves in flasks were transferred to soil in field. A protocol has been development for in vitro rooting and hardening and en vitro transplant of Podophyllum hexandrum Royle. Roots were efficiently established on WPM media supplemented with IAA 1.5 mg·L^-1 and NAA 0.5 mg·L^-1. Before transferred to soil containing turfysoil and perlite (2:1), rooted plants were exposed to air for 5d for adaptation. Two periods were good for transplanting this plantlets.