Description of the pharmacological activities of Sanghuang mushrooms (Inonotus Sanghuang) can be traced back to Tang dynasty of China 1300 years ago. This mushroom has been widely accepted in China, Japan, Korea and certain regions of Europe as a nutraceutical medicine for enhancing immunity or an alternative medicine for prevention or inhibition of tumorigenesis. However, this mushroom is rarely available from the mulberry trees in the wild because of the rigorous conditions needed for formation of the Sanghuang mushrooms.This study aims to establish a practical protocol for culture, particularly for a bunch of production of Sanghuang mushrooms possibly to commercialize the cultured Sanghuang based on deep comparison of quality and pharmacological activities between the cultured and the wild Sanghuang.A phylogenetic tree containing five strains of the wild Sanghuang was constructed using rDNA markers. Different temperatures and medium compositions were surveyed to develop a practical protocol for culture of the Sanghuang mushrooms. 5-fluorouracil was used to induce the immunodeficient mice. Chemotherapeutic components and pharmacological activities were deeply analyzed between a cultured strain (SG) and three strains of the wild Sanghuang.Maintenance of a temperature of 22-28 °C and a high relative humidity of 90-95%, and use of a high ratio (80%) of mulberry tree sticks in the medium were critical to successful culture of Sanghuang. The cultured mushrooms were yellow with a uniform shape, while the wild Sanghuang was dark brown with a smaller and irregular shape. The cultured mushrooms contained significantly higher levels of polysaccharides, amino acids, and water-soluble nutraceuticals, whereas flavones in the wild Sanghuang were significantly higher (P < 0.05). Use of a dose of 8 mg/kg or 16 mg/kg to immunoregenerate the immunodeficient mice was comparable between the cultured and wild Sanghang based on analysis of hematological parameters and histological examination of the thymus and spleen in the treated mice.This study highlights the potential of the immunoregenerative functions of the cultured Sanghuang for cancer chemotherapy and suggests that the cultured Sanghuang can be an alternative to wild Sanghuang used for nutraceutical medicine.