Abstract: The underground buds of Paeonia lactiflora ‘Zhong Sheng Fen’ were used to study various concentration of IAA and sampling time on initiation culture, various concentration of 6-BA on axillary shoots inducing, and different auxin on axillary shoots proliferation and rejuvenation of hyperhydric plantlets. The results showed that: the best initial medium of ‘Zhong Sheng Fen’ is 1/2 MS(double Ca2+)+ 6-BA 1.0 mg·L-1 + GA3 0.5 mg·L-1+IAA 0.1 mg·L-1. The highest germination percentage (92.80%), number of lateral shoots (2.13) and height (4.22 cm) were observed in the treatment. It was better to sample underground buds in spring than in winter. The germination percentage was 100.00% in spring and 96.70% in winter. Leaf expansion percentage was 92.60% in spring and 61.5% in winter. 2.0 mg·L-1 6-BA was good for axillary shoots inducing. 5.67 axillary shoots were induced from the main buds. In addition, the lowest death percentage (6.67%) was gained. Auxin was not good for axillary shoots proliferation. Adding AC and removal ammonium nitrate in the medium, using triangular flask as culture vessel and joint filling material, increasing calcium (double Ca2+) in the medium and not using 6-BA were helpful to the rejuvenation of hyperhydric plantlets. The percentage of normal shoots can reach 20.06%-34.37%.