Cremastra appendiculata (D. Don) Makino (Orchidaceae) is a valuable species which is in ornamental field and pharmaceutical industry, inefficient propagations limit the utilization of C. appendiculata. Here, we reported an improved and efficient method for large-scale micropropagation including seed germination, initiation and proliferation of protocorm like-bodies (PLBs), “shoot cluster” induction, rooting and acclimatization. Factually, the highest shoot multiplication coefficient 3.16 was achieved on 1/4 MS medium supplemented with 1.5 mg·L -1 thidiazuron (TDZ), along with 0.4 mg·L -1 indole-3-acetic acid (IAA), 3% sucrose and additives. Likewise, the best medium for protocorm proliferation was 1.0 mg·L -1 indole-3-butyric acid (IBA), 1.0 mg·L -1 6-benzyladenine (6-BA) and 0.2 mg·L -1 adenylate cyclase (AC) with proliferation of 170.07%, followed by root induction on 1/4MS with 0.5 mg·L -1 α naphthaleneacetic acid (NAA), 0.5 mg·L -1 AC and 3% sucrose. Besides, the achieved micropropagation plants grew vigorously and were steady with a survival rate of 85.3% in vermiculite: humus (1: 2). Assessment of the chemical compositions by high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) displayed that the composition of dried pseudobulbs from micropropagation plantlets and naturally grown plants were similar, containing colchicine, quercetin, rutin, adenosine, oleanolic acid, myristate, rhein and emodin. Furthermore, the hypoglycemic activity in vitro indicated micropropagation plantlets were superior to naturally grown plants. Consequently, the described protocol could be used for the large-scale propagation and conservation of this endangered plant, which might be natural resource for the diabetes patients.