Abstract: Glucose phosphate isomerase (PGI) catalyzes the conversion of 6-phosphate-glucose to 6-phosphate-fructose, which is an important branch node in the process of polysaccharide synthesis. By using PGI protein of Ganoderma lucidum as a probe, based on the genome database of Antrodia cinnamomea, the pgi gene of Antrodia cinnamomea was cloned by using the electronic cloning technology, and the characteristics of the protein encoded by the gene were analyzed. The transcript expression level of the gene during the fermentation process was detected by RT-PCR. The results showed that the cDNA length of pgi gene was 1 659 bp, encoding 552 amino acids, and the encoded PGI protein was hydrophilic and had no transmembrane and signal peptide structure. The secondary structure was dominated by 47.83% α-helix. The protein sequence had a close genetic relationship with Sparassis crispa and Amylocystis lapponica. In the process of solid culture and liquid culture of Antrodia cinnamomea, the level of pgi gene transcription expression increased with time, which was inversely proportional to the synthesis of polysaccharides. The results of this study could lay a foundation for the metabolic regulation of polysaccharide synthesis in Antrodia cinnamomea.