One way to reduce the energy content of foods while maintaining the sweet taste is the application of non-caloric sweeteners. However, knowledge regarding the consequences of a long-term exposure to sweeteners on gene expression of sweet sensing pathways in the human intestine is scarce. Here we investigated the impact of three common non-caloric sweeteners in comparison to sucrose in equi-sweet and equi-molar physiologically relevant concentrations on gene expression of nutrient-sensors in cell culture models of the small intestine. A coculture model of Caco-2 enterocytes and mucus-producing HT29-MTX-E12 goblet cells in comparison to Caco-2 cells in monoculture was established to obtain a physiologically more relevant model. Both cell models were treated during differentiation with either sucrose, rebaudioside M, sucralose, or neohesperidin dihydrochalcone for 7, 14, or 21 days, and the gene expression of TAS1R3, SLC2A2, SLC5A1 and KCNJ8 was analyzed by qRT-PCR. The coculture model showed a physiologically more relevant transepithelial electrical resistance and mucus production than the monoculture, in addition to an in vivo-like paracellular permeability. Treatment with the sweeteners led to less pronounced regulation of the target gene expression in the coculture model. The treatment with sucrose applied in an equi-sweet concentration to the non-caloric sweeteners had the strongest impact on the expression of the target genes, e.g. a 1.44 ± 0.05 -fold (p < 0.0001) increase in SLC5A1 expression was detected after 14 days of treatment with sucrose, but not the other sweeteners in Caco-2 cells. The results further indicate that there was no impact of the sweet intensity on the expression of the marker genes. In conclusion, structure-specific effects for sweeteners on gene expression of nutrient sensing pathways were demonstrated in the cell models for the human intestine, with stronger effects in the Caco-2 monoculture than in the coculture model using goblet cells.