Cypripedium guttatum is globally threatened species due to over-collection from the natural habitats for horticultural purpose. It is very rare in nature and is, therefore, one of the most endangered species. Establishment of an efficient method for in vitro germination of seeds is urgently needed. In this paper we report the propagation of C. guttatum via in vitro seed germination of fully mature seeds. Treatment with 1% NaOCl greatly enhanced the frequency of swelling and protocorm formation from zygote embryos of seeds compared to control. Shoot and root formation from protocorms was better in Phytomax Orchid Maintenance (POM) medium than Murashige and Skooge (MS) medium. Growth of roots from protocorms was stimulated on medium containing 14.43 μM GA₃ and 0.2% activated charcoal (AC). Cold treatment was necessary for shoot growth from protocorms. Plantlets were transferred to soil after cold treatment for three months. The converted plantlets were acclimated in artificial soil and then transferred to greenhouse. The results obtained appear to be applicable to plant propagation of C. guttatum via asymbiotic germination and growth.