Abstract: The manganese superoxide dismutase of Pleurotus tuber-regium was extracted and identified. The manganese superoxide dismutase (Mn-SOD) gene of P. tuber-regium was cloned from the mycelium of P.tuber-regium. Mn-SOD of P.tuber-regium was extracted by liquid nitrogen grinding, ultrasound, ammonium sulfate precipitation and dialysis. Mn-SOD activity of P. tuber-regium was determined by WST-8 method. SOD type of P. tuber-regium was identified by H₂O₂. Mn-SOD gene was cloned from P. tuber-regium by homologous cloning, rapid amplification of cDNA ends, fusion primers and nested PCR. Results showed that the activity of Mn-SOD of P. tuber-regium was 1.66 U. There was no significant difference between the enzyme treated with H₂O₂ and untreated, indicating that the main SOD in P. tuber-regium was Mn-SOD. PtMn-SOD was cloned and obtained. Its DNA sequence was 1025 bp in total, 663 bp in open reading frame and 220 amino acids were encoded. Sequence analysis and phylogenetic tree showed that PtMn-SOD was closely related to Pleurotus ostreatus (accession number:MH645359.1). It was predicted that the molecular weight of the protein was 24.54 kDa and the isoelectric point of the protein was 7.86. PtMn-SOD protein located in mitochondria and played a role in mitochondria.