[Objective] The study aims to explore the in vitro regeneration conditions of Codonopsis pilosula (Franch.) Nannf. and establish its artificial efficient rapid propagation system, to provide a theoretical basis for its seedling breeding. [Methods] The callus was induced by the stem tip and stem segment in different exogenous hormone combinations, and then the L16 (45) orthogonal experiment was carried out with coconut juice, 6-BA, NAA and KT to screen out the best explant and the differentiation and proliferation medium of callus-adventitious buds. Finally, the rooting and acclimatization of plantlets were realized outside the bottle. [Results] (1) The callus induction rate of stem segments in MS medium with 6-BA of 1.5 mg/L and NAA of 0.5 mg/L was up to 98.76%, while that of stem tips was only 76.56%. (2) Coconut juice had a significant effect on the proliferation of C. pilosula, and the proliferation coefficient reached 43.39 in MS medium with coconut juice of 60 mL/L, 6-BA of 1.5 mg/L, KT of 0.5 mg/L and NAA of 0.5 mg/L. (3) The rooting rate of regenerated buds in bottles was very low, but the regenerated buds rooting outside the bottle could form roots after 30 days, with a rooting rate of 87.96%, and the survival rate after transplanting was 97.73%. In addition, basal stem cluster buds and axillary buds appeared during callus induction and differentiation, which also greatly improved the proliferation coefficient of C. pilosula. [Conclusion] The stem segment was more suitable for callus induction, coconut juice promoted callus redifferentiation, and rooting outside the bottle significantly improved the rooting rate.