The sodium/glucose co-transporter 1 (SGLT1) is primarily responsible for the intestinal uptake of glucose through an active transport driven by the Na+/K+ ATPase. Homozygous mutation of the gene encoding SGLT1 (SLC5A1) are the cause of glucose/galactose malabsorption. It has recently been shown that carriers of a haplotype with 3 missense mutations (N51S, A411T, and H615Q) of SLC5A1 exhibit lower 2-hour glucose levels during OGTT and a lower risk of impaired glucose tolerance. Subjects with normal glucose levels at fasting and 2h-OGTT (normotolerant, NGT) but with 1h-OGTT ≥155 mg/dL (NGT-1h-high) have a worse cardio-metabolic risk profile and higher incidence of type 2 diabetes compared to the NGT population with 1h-OGTT glucose <155mg/dl (NGT-1h-low). In this study, we investigated whether the N51S/A411T/H615Q haplotype of SGLT1 was associated with 1h-OGTT glycemic pattern in NGT subjects, and we assessed the functional significance of these variants in vitro. 800 well-characterized adult NGT subjects were subjected to a 75g OGTT and stratified into two groups: NGT 1h-high or NGT 1h-low. The frequency of the rare allele of each SNP was higher (p<0.05) in NGT-1h-low subjects (1.515% N51S, 9.139% A411T, 3.926% H615Q) than in NGT-1h-high subjects (0.549%, 1.315%, 1,515%, respectively). When the 3 SNPs were collectively analyzed in a genetic risk score, it was observed that carriers of rare alleles had a dose-dependent lower risk of showing 1h-OGTT glucose levels ≥155 mg/dL (OR 0.419, CI95% 0.217-0.810, p<0.01). Site specific mutagenesis was performed in order to introduce the three variants into a cellular model of human intestinal epithelium (CACO2 cells). CACO2 cells were transfected for 48 hours to overexpress comparable levels of wild-type and mutant isoforms of SGLT1. Glucose uptake (measured by commercial kit) was significantly reduced (p<0.0001) in CACO2 cells expressing mutant SGLT1 (%reduction=~29% N51S, ~30% A411T, ~33% H615Q). CACO2 cells carrying any combination of double or triple mutations showed an even stronger reduction of their glucose transport ability compared to wild-type (37-49%, p<0.0001. These data suggest that the genetic variants of SGLT1 may have a role in the determination of early post-prandial glycemic patterns. This research might drive the development of novel therapeutic approaches targeting intestinal glucose uptake with the intent of preventing the formation of a dysglycemic milieu, and long-term cardiometabolic protection.