Glyphosate, a widely used herbicide in North America, has become prevalent in the food supply, raising concerns about potential health impacts. In this exploratory study, male and female F0 mice were exposed to glyphosate through drinking water during mating and gestation. We investigated whether prenatal exposure at dietary-relevant levels (0.01 mg/kg/day, Average American Diet, [AAD]) or the U.S. EPA's acceptable daily intake (1.75 mg/kg/day, [EPA Upper Limit]) altered gut, metabolic, and behavioral outcomes across two generations in mice with or without genetic susceptibility to colitis (Muc2and Muc2, respectively). Healthy (Muc2) offspring of glyphosate-exposed mice exhibited colonic goblet cell depletion, reduced mucin-2 expression, and pro-inflammatory cytokine profiles in both F1 and F2 generations. These healthy (Muc2) offspring also developed metabolic dysfunction, including impaired glucose tolerance, insulin resistance, and reduced GLP-1 in serum. Behavioral deficits were also observed in healthy (Muc2) mice including reduced locomotion and working memory. These changes were accompanied by significant shifts in gut microbiome composition and associations between specific microbes, including Akkermansia muciniphila and Parabacteroides distasonis and gut-brain signaling molecules including GLP-1 and serotonin, suggesting a microbiota-mediated mechanism for neuroendocrine disruption. In contrast, colitis-susceptible (Muc2) mice showed fewer overt exposure effects, likely due to masking by their baseline disease phenotype, but did show alterations consistent with enteric neuroinflammation resulting from glyphosate exposure including blooms in Cyanobacteria and reduced tryptophan metabolites. These findings suggest that prenatal glyphosate exposure, even below regulatory thresholds, may disrupt multiple physiological systems across generations, highlighting the need for further research and potential regulatory consideration. +/--/-+/-+/-+/--/-
