To establish a stable and highefficiency plant regeneration system for Pterocephalus hookeri (C.B.Clarke) Heck via plant tissue culture, we studied the optimum seed disinfection method, and the main factors which affected callus induction, proliferation and plant regeneration of explants from aseptic seedling. The results indicated that: (1) the optimum disinfection method for seeds removed exopleura of P. hookeri was 75% ethanol 1 min＋0.1% HgCl₂ 7 min＋derosal (50%wp, diluted 500 times)30 min, the seeds were thoroughly bioclean, and the germination rate was 60.67%. (2) Leaf piecesand stem segments excised from the seedlings were suitable to induce calluses with the percentage of 84.00% and 97.33% respectively on medium MS＋5.0 mg·L^-1 6BA＋2.0 mg·L^-1 2,4D after 10 d. (3) The optimal medium for callus proliferation was MS＋3.0 mg·L^-1 6BA＋2.0 mg·L^-1 IAA, growth rate of calluses reached 74.37% and 70.52% with leaf pieces and stem segments, respectively. (4) The optimal medium for clustered shoots induction was MS＋3.0 mg·L^-1 6BA＋2.0 mg·L^-1 IAA＋250 mg·L^-1 Lproline＋150 mg·L^-1 casein hydrolysate, the differentiation rate were 100% and 94.44% with leaf and stem, respectively. (5) The optimal rooting medium was 1/2 MS＋0.5 mg·L^-1 NAA, the rooting rate was 100% after 30 d. As a result, an efficient plant regeneration system was established in order to provide an effective solution for protecting the wild resources and industrial seedling of P. hookeri.