A single‐stage batch in vitro fermentation system has been designed to simulate distal colon microbial activity. This system allows for examination of bacterial function. Outputs include filtered fecal supernatant (FS) that can be used for metabolite identification and examining host response. The goal of this study was to treat human duodenal and colonic enteroids with FS to determine how microbial metabolites alter cell proliferation, differentiation, and expression of other intestinal factors. Three‐dimensional (3D) human duodenal and colonic enteroids were treated with 0, 0.1, 1, 10, 25, and 50% FS or 1μg/mL lipopolysaccharide for 24 h. Cell lysates were used for RNA extraction and mRNA analysis. Visually, increasing FS concentration caused increased browning of enteroids as signs of cell differentiation. At 25 and 50% FS, structural disruption occurred, with fargreater effect at 50% potentially indicating cell death. Expression of LGR5, a stem cell marker, decreased with FS concentration and was lowest at 10% (P < 0.01). EdU staining confirmed this as cell proliferation was significantly decreased when treated with concentrations >1% FS. Expression of enterocyte and Paneth cells markers were increased by greater than 14‐fold at the 10 and 25% concentrations for alkaline phosphatase (ALPI), and six‐fold at 25% for lysozyme (LYZ), respectively (P < 0.01). Mucin 2 (MUC2) expression was increased by eight and 50‐fold at the 10 and 25% concentrations (P< 0.01) respectively, and two and three‐fold increases for mucin 3a (MUC3A; P < 0.01) suggesting an overall increase in goblet cell number. Expression of E‐Cadherin (ECAD), occludin(OCLN), claudin‐3 (CLDN3), and tight junction protein 1 (TJP1) were increased two to four‐fold and six to 23‐fold in the 10 and 25% FS groups respectively (P < 0.01). In a two‐dimensional duodenal enteroid monolayer grown on transwell inserts, there was a 13 and 25%increase in transepithelial electrical resistance (TER) at 10 and 25% FS concentrations respectively over a 24 h period (P< 0.01), and a three‐fold increase after 72 h with 25% FS (P < 0.01). In 3D colonic enteroids treated with FS there was a reduction in LGR5 expression while ALPI and LYZ were upregulated ten and three‐fold, respectively, at the 25% FS concentration. Expression of MUC2, MUC3A, Regenerating islet‐derived protein 3 alpha (REG3A), and trefoil factor 3 (TFF3) were substantially increased (>8‐fold) in the 25% FS group (P < 0.01), although in the10% FS group increases were two to three‐fold (P< 0.01) and TFF3 was not different from other treatments. The 10% FS group also saw an increase in ECAD and TJP1 expression while CLDN3 was decreased (P < 0.01). Interestingly, the 25% FS group saw a two to four‐fold increase in ECAD, OCLN, CLDN3 and TJP1 (P < 0.01). Overall, treatment with FS alters enteroid cell differentiation, and expression of mucin and tight junction protein mRNA, likely similar to a typical intestinal phenotype.Support or Funding InformationDepartment of Defense Combat Feeding Research and Engineering Program and The Applied Research for the Advancement of Science and Technology Priorities (ARAP) Program on SyntheticBiology for Military Environments (SBME), through the Office of the Secretary of Defense.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.