Glucose is a critical nutrient for energy metabolism. The SLC2A2 gene is essential for glucose sensing and homeostasis, as it encodes the facilitated glucose transporter GLUT2. During diabetes treatment, the C-allele of rs8192675 in SLC2A2 has been found to regulate the action of metformin and reduce the absolute level of HbA1c more effectively than the T-allele. In this study, stable HEK293T cell lines carrying the CC, CT, and TT genotypes of rs8192675 in SLC2A2 were generated using CRISPR/Cas9-mediated genome editing. GLUT2 mRNA and protein levels were elevated in cell clones with the TC genotype compared to those with the CC genotype but were reduced relative to the TT genotype. Additionally, high concentrations of glucose or fructose induced more GLUT2 protein production in CT-genotype cells than that induced in CC-genotype cells, yet less than that induced in TT-genotype cells. Metformin induced a greater increase in GLUT2 expression and a smaller increase in activated AMPK protein expression in CC-genotype cells than those induced in TT-genotype cells, resulting in a remarkable reduction in activated mTOR and S6 levels. This study directly supports the biological mechanism linking the C-allele of rs8192675 with improved treatment outcomes in metformin therapy for diabetes.