Abstract: Weissella cibaria M165 with a mulberry fruit wine histamine degradation ability was selected as the research object, and the key histamine degradation enzyme of W. cibaria M165 was revealed. The potential key histamine degradation enzyme gene (glyceraldehyde 3-phosphate dehydrogenase gene) of W. cibaria M165 was obtained by whole genome sequencing of W. cibaria M165. Meanwhile, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was confirmed as the key histamine degradation enzyme of W. cibaria M165 by recombinant expression and histamine degradation analysis. Results showed that, the degradation rate of histamine in mulberry fruit wine by GAPDH was 31.88%, and it had no negative effect on the content of active substances in mulberry fruit wine. In order to further analyze the key action sites for GAPDH degradation of histamine in mulberry fruit wine, the potential key action sites for GAPDH degradation of histamine in mulberry fruit wine were screened by molecular docking (Ser 125, Cys 155, Phe 322), and these sites were mutated to alanine. Compared with unmutated GAPDH, the enzyme activity and histamine degradation rate of the mutant proteins were significantly increased (P<0.0001), among which the enzyme activity and histamine degradation rate of GAPDH-C155A and GAPDH-F322A were more changed. The enzyme activity of GAPDH-V241A and GAPDH-I286A was increased by 62% and 97%, respectively. Moreover, the histamine degradation rate of GAPDH-C155A and GAPDH-F322A was enhanced by 31.51% and 39.58%, respectively. Above results indicated that Cys 155 and Phe 322 were the key action sites for GAPDH degradation of histamine in mulberry fruit wine, which could be used as the modification targets for the enhancement of histamine degradation ability of GAPDH.