Ardisia gigantifolia Stapf is native to Hekou county, Yunnan province, with few wild individuals, and is listed as a critically endangered species. It has important protection value and good prospect of garden development and application. The stem segments of aseptic seedlings of A. gigantifolia were used as the explants to establish a propagation in vitro. The results showed that the optimal medium for callus induction was WPM+0.50 mg/L TDZ+0.10 mg/L NAA, and the induction rate of callus was 96.67%. The optional medium for callus proliferation was WPM+0.50 mg/L TDZ and the multiplication coefficient was 4.42. The optimal medium for callus differentiation was WPM+2.00 mg/L 6-BA+0.20 mg/L NAA, and the induction rate reached 100%. The optional medium for adventitious bud proliferation culture was WPM+4.00 mg/L 6-BA, the multiplication coefficient was 3.90, and the cluster buds grew well. The optional rooting medium was WPM+10% coconut water+0.20 mg/L NAA, the rooting rate reached 100%, the root system developed well and the test-tube plantlets grew vigorously. Plantlets were transplanted into the mixture substrate with the volume ratio of perlite and peat soil of 1∶3, the survival rate was 96.67% after 60 days and plants grew well. This study can provide technical support for the large-scale seedling production of A. gigantifolia, as well as theoretical basis and technical support for the protection, seedling breeding, and molecular biology research of this species.