Objective: To investigate the serological characteristics and molecular mechanism underlying weakened B antigen expression caused by a c.28G>A (p.Gly10Arg) mutation in exon 1 of the ABO gene. Methods: Peripheral blood samples were collected from a proband and his parents. ABO phenotyping was performed using the microcolumn agglutination and saline tube methods. Sanger sequencing of exons 1-7 of the ABO gene was conducted to identify the mutation. Comprehensive bioinformatics analysis was performed using Jalview, PolyPhen-2, ESEfinder, RNAfold, TMHMM, and IUPred2A to assess evolutionary conservation, pathogenicity, splice regulation, RNA structure, transmembrane topology, and disordered regions. Results: Serological tests identified the proband and his mother as A?Bweak, while the father was A?. Sequencing revealed both the proband and mother carried ABO*A1.02/B.01 alleles, with a heterozygous c.28G>A mutation on the B* allele. Pedigree analysis confirmed co-segregation of the mutation with the phenotype. Evolutionary conservation analysis showed the Gly10 residue is highly conserved across multiple mammalian species. Bioinformatics prediction indicated the mutation was "benign", but potentially created a new SC35 splicing factor binding site. It did not alter the minimum free energy of the local RNA secondary structure. While the transmembrane topology remained unchanged, the ANCHOR score for interaction propensity slightly increased. Conclusion: The c.28G>A missense mutation in exon 1 of the ABO gene leads to weakened B antigen expression and the A?Bweak phenotype. This mutation occurs at an evolutionarily conserved site. Its molecular mechanism is primarily associated with the creation of an aberrant splicing enhancer that disrupts RNA processing, rather than directly impairing protein structure and function.