Artificial sweeteners (AS) are synthetic sugar substitutes that gained popularity for their low-price and for no-calorie gain. However recent studies have demonstrated considerable health risks since AS consumption has been linked to metabolic derangements and gut microflora perturbations (Suez et al., 2014). Several studies have represented AS consumption-related intestinal barrier impairment and gut bacteria dysbiosis. But, how AS affect the intestinal epithelial cell function or change the commensal bacteria to pathogenic has not yet been defined. The current in vitro study investigated the effect of physiologically-achievable concentrations of three commercially-available AS on the monolayer permeability of intestinal epithelial cell model, Caco-2 cells and metabolism of model gut bacteria, E. coli NCTC 10418. Also, the pathogenic potential of the bacterium in terms of biofilm formation and haemolysing red blood cells was studied. In addition, AS (100 µM) effect on the bacterial adhesion and invasion ability to the intestinal epithelial cells was assessed. Data were analyzed using GraphPad Prism (v.8), compared by ANOVA and values are presented as mean ± S.E.M. Saccharin and sucralose at physiological concentration (1 mM) significantly increased intestinal epithelial cell monolayer permeability percentage (195±34 and 164±18, respectively) whilst aspartame was found to increase leak (377±90) at sub-physiological concentration (100 µM) (p<0.05, n=5). There observed no effect of the three sweeteners (100 µM) on the model bacteria growth and no change in its characteristic α-hemolysis pattern. However, all the tested sweeteners significantly increased (almost doubled) the biofilm formation (n=5) and inhibiting the sweet taste sensing reduced the effect. E. coli exposed to sucralose and aspartame showed a significant increase in their adhesion/invasion percentage (204±26 / 267±61 and 286±66 / 249±58, respectively) to Caco-2 cells, however, saccharin exposure significantly increased adherence (226±41) but not invasion (107±26) (p<0.05, n=4). Similar to biofilm formation, exposure in the presence of zinc sulfate attenuated the bacterial pathogenic effect. These data suggest that AS causes a leak in the intestinal epithelium, which can lead to inflammatory responses. Also, AS exposure induces bacterial pathogenic factor and promotes their adherence and intrusion to intestinal epithelial cells. Further investigations on the mechanisms through which AS affect gut bacteria and bacteria-epithelium interactions will extend our understanding of sweetener-induced dysbiosis formation. Also, the mechanism of how zinc sulfate exerts an inhibitory effect on model bacterial pathogenicity needs to be investigated. Since artificial sweetener consumption in the diet continues to increase, it is vital to understand how this food additive affects microflora and gut health. Keywords: Artificial sweetener; microflora; dysbiosis; epithelial cells.