Glucose is one of the nutritional factor that involves in developing of obesity and type 2 diabetes in human. The studies indicated that enterocyte cells on intestine might play a role in dietary glucose sensing during obesity. Obese people are consumed high amount of dietary glucose and enterocyte cells consequently are exposed to high glucose. Thus, we aimed to find relevant physiological pathways and genome-wide mRNA expression profiles that can be regulated by glucose in fully differentiated human intestinal epithelial (CaCo-2). The cells were maintained two different glucose levels (5.5mM for control, 25mM for high glucose) at least three passages. The cells were grown on transwell system for 21 days to mimic human intestine system. Transepithelial electrical resistances (TEER) were measured to control monolayer formation and polarization. RNA isolation was performed and whole genome mRNA expression profile were determined following gene ontology analysis to find affected molecular pathways. Compared to control relative glucose level was found high in basolateral side of CaCo-2 cells that were under high glucose condition without effecting TEER. GLUT2, SGLT1, GLUT5 mRNA levels were significantly reduced during elevated glucose levels which is consistent with literature. Significant fold change analysis showed that 351 genes upregulated and 468 genes under high glucose condition. We found high glucose significantly leads changes of molecular pathways (downregulated; glycolysis and gluconeogenesis, adherens junction, fructose/mannose metabolism, pentose phosphate pathway and upregulated; protein export). These results provide us better understanding and open new window for glucose metabolism of enterocytes during obesity.