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The rise in popularity of non-caloric sweeteners (NCS) among obese and diabetic patients is due to their ability to provide a sweet taste without any caloric input. However, NCS consumption may be linked to metabolic disorders. The mechanisms behind these adverse effects are still unclear, which suggests that NCS may alter the metabolic activity of the gut microbiome, leading to enteric environmental perturbation and resulting in physiological anomalies in the host. This study investigates how Escherichia coli, a common gut microbe, reacts to three NCS, two of which come from nature (glycyrrhizin and stevioside) and one that is made artificially (sucralose). To assess the impact of these NCS, this study investigated the growth, gene expression, and metabolic pathways of E. coli under different sweetener loads. Compared to the untreated control, low amounts of glycyrrhizin made E. coli grow faster, while similar amounts of sucralose and stevioside had no or a less noticeable effect. At higher concentrations, these sweeteners slowed down or inhibited the growth of the bacteria. Thus, NCS may have dose-dependent impacts on gut microbes. After treatment with amounts that corresponded to the acceptable daily intake, the expression of key metabolic genes showed altered expression, which may potentially affect their metabolism. At different concentrations of the NCS, there were clear differences in the global metabolomic profile between the control and the different NCS treatments. Under in vitro conditions, glycyrrhizin and sucralose appeared to have a less divergent impact on E. coli growth, gene expression, and metabolism, which may suggest that these are relatively safer sugar substitutes for humans. [ J Adv Biotechnol Exp Ther 2023; 6(3.000): 659-672]