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. Based on the genome database of Antrodia cinnamomea, the PGI gene of A. cinnamomea was cloned by using the PGI protein of Ganoderma lucidum as a probe. The characteristics of the protein encoded by the gene were analyzed, and the transcriptional expression level of the gene during fermentation was detected by RT-PCR. The cDNA length of the gene is 1659 bp, encoding 552 amino acids. The encoded protein is hydrophilic and has no transmembrane and signal peptide structure. The secondary structure is mainly 47.83% α-helix. It has a close genetic relationship with Spirulina in molecular evolution. In the process of solid culture and liquid culture of A. cinnamomea, the level of gene transcription expression increased, which was inversely proportional to the synthesis of polysaccharides. The study laid a foundation for the metabolic regulation of polysaccharide synthesis in A. cinnamomea.