Abstract: The regeneration plants were acquired by culturing in vitro the growing points (in 0.2-0.5 mm length) of Pseudostellaria heterophylla.The orthogonal experiments and single factor experiments showed that the optimum medium for calli induction and shoot buds from calli was MS+6-BA 2 mg·L-1+NAA 0.2 mg·L-1 + GA30.1-0.2 mg·L-1+sugar 30 g·L-1.The medium suitable for calli subculture was MS+6-BA 1.5 mg·L-1 + NAA 0.01 mg·L-1+sugar 30 g·L-1.The plantlets’propagation had been done by 2 ways i.e.continuous calli differentiation and plantlets cuttage propagation.Basic MS medium was suitable for plantlets cuttage propagation.Medium MS (pH 6.0) containing 60 g·L-1 sugar was suitable to induce micro-root tuber, under the condition of 20℃ and 16 hours of light everyday.Medium MS containing 30 g·L-1 sugar was suitable for roots differentiation.The survival rate of the tissue culture plantlets exposed to air for 10-15 days reached 98.6% in the sandy soil under the condition of 15-20℃ and the relative moisture more than 90%.The mosaic disease rate of virus-free roots was 94.75 % lower than that of conventional culture system in the field.