Antrodia cinnamomea is a unique medicinal fungus in Taiwan. It has been found rich in some pharmacologically active compounds for anti-cancer, hangover, and immune regulation etc. With the in-depth study of these components, it would be interesting and important to establish a molecular system for basic studies of A. cinnamomea. Thus, we would like to set up a foundation for this purpose by studying the A. cinnamomea protoplast preparation and regeneration. Firstly, we studied the optimization method of protoplast preparation of A. cinnamomea, and found various factors that may affect the yield during protoplast preparation, such as mycelial ages, pH values, and osmotic stabilizers. Secondly, in the regeneration of protoplasts, we explored the effects of various conditions on the regeneration of protoplasts, including different media and osmotic pressure. In addition, we found that citrate buffer with pH value around 3 dramatically increased the regeneration of protoplasts of A. cinnamomea, and provided a set of regeneration methodology for A. cinnamomea.