Objective · To analyze the effect of glucose-6-phosphate dehydrogenase (G6PD) gene mutations on the structure and function of enzymes. Methods · A retrospective analysis of G6PD screening records of 205 103 neonates collected in the Shanghai Children's Hospital from 2014 to 2017 was performed. Wilcoxon test was used to analyze the enzyme activity and mutant genes. The software tools Psipred, SOPMA, and JPred4 were used to predict the secondary structure of the protein, and the 3D structure of G6PD was predicted based on the amino acid chain by SWISS-MODEL and modified by PyMOL and LigPlot+. Six different analysis software programs, including Mupro, SDM, CUPSAT, mSCM, DUET, and Dynamut, were utilized to compare protein stability of the wild-type with the mutant forms. PROVEAN was used to analyze the effect of amino acid changes on the enzyme. Results · Two hundred and thirty samples were positive for enzyme deficiency, of which 121 positive samples were genetically tested and eight mutations were identified. Three common mutations c.95A>G, c.1376G>T, and c.1388G>A were present, and c.1024C>T mutation was identified in 14 samples. These four mutations induced a change in protein structure, reduced protein stability, and had adverse effects on function. In addition, c.1388G was adjacent to the glycerol ligand binding region in the wild type structure, whereas the c.1388G>A mutation caused this residue further away from it. Conclusion · Four nonsynonymous mutations (c.95A>G, c.1376G>T, c.1388G>A and c.1024C>T) reduce the stability of the G6PD enzyme by changing the structure of the protein, in which the c.1388G>A mutation also affects the binding ability of the protein to the substrate.