Abstract Disclosure: A.T. Coren: None. M.F. Roethle: None. L. Zhang: None. A.Y. Pan: None. S. Jia: None. M.J. Hessner: None. S.M. Cabrera: None. While genetic predisposition for type 1 diabetes (T1D) is strongly conferred by HLA-DR3 and/or HLA-DR4, shifting environmental factors that potentiate β-cell autoimmunity are believed responsible for increasing T1D incidence. Evidence suggests that intestinally mediated exposures, such as the Western diet, influence immunity and T1D risk. Gluten is a potential mediator of inflammation. We have described endogenous innate inflammation in individuals with T1D and their healthy siblings and that T1D progresses when this innate state is augmented by additional inflammatory signals prior to development of protective counter-regulatory mechanisms. We hypothesize that, in genetically susceptible hosts, gluten promotes the innate inflammation that potentiates T1D progression and that its removal will abrogate this risk. In an exploratory pilot study, we enrolled healthy children who had a full sibling with T1D to a 4-wk gluten free diet (GFD). Adherence was measured by self-report and fecal gluten peptide. Pre- and post-GFD plasma was analyzed for changes to microbial antigen detection by TLR4 activation, short chain fatty acids (SCFAs), cytokines, and chemokines. Wilcoxon signed rank tests were used to examine paired data. Stool microbial composition was measured by 16s rRNA bacterial gene sequencing and analyzed by generalized linear mixed models with negative binominal distribution. P<0.05 was considered significant. 28 participants completed the study (mean 13 years, range 2.7-22.1, 20 (71%) females, 22 (79%) non-Hispanic white, 9 (32%) pre-pubertal). 21% had a first degree relative with celiac disease and 11 (39%) had HLA-DR3 and/or DR4. At baseline, 50% had inadequate dietary fiber intake. There was no attrition, but 29% admitted to occasional gluten consumption or had detectable fecal gluten peptides. TLR4 activity trended lower post-GFD (p =0.06). Post-GFD, plasma acetate, an anti-inflammatory SCFA, was increased; hexanoate, an inflammatory fatty acid, was decreased (p<0.001). The plasma-to-fecal ratio of acetate, propionate, butyrate, valerate, and hexanoate were increased. Several inflammatory cytokines/chemokines were significantly reduced post-GFD (IFNα, IL-7, MCP-1, CCL5, Eotaxin-2) but IL-1α and TNFβ increased. Fecal 16s rRNA analyses revealed no GFD-driven differences in alpha or beta diversity, but at genus level, post-GFD samples had lower abundances of Roseburia, Faecalibacterium, and Bifidobacterium and higher abundances of Bacteroides. In sum, removal of gluten reduced several markers of systemic inflammation, independent of HLA-DR3/DR4, suggesting gluten may potentiate innate inflammation and T1D progression in those with underlying susceptibility. This may occur by altering gut barrier integrity and/or shifts in microbial taxa/metabolites. These data suggest mechanisms by which gluten may directly and indirectly drive T1D progression. Presentation: Thursday, June 15, 2023