Irritable Bowel Syndrome (IBS) is a disorder of the gut-brain axis, with altered gut function and frequent psychiatric co-morbidity. We have previously shown in gnotobiotic mice that fecal microbiota from patients with IBS with diarrhea (IBS-D) and co-morbid anxiety induces faster gastrointestinal transit, gut barrier dysfunction, immune activation and anxiety-like behavior. However, the exact mechanisms underlying these abnormalities are not understood. To investigate and characterize the role of neural and immune factors in the microbiota-mediated alteration of gut physiology and behaviour. The expression of 72 murine genes related to neural, immune, and epithelial function was measured with NanoString nCounter® gene assay on total RNA extracted from colonic sections of mice colonized with microbiota from IBS-D patients or healthy volunteers. Colonic histology sections were stained for F4/80+ cells and multi-parameter flow cytometry was used to survey population of conventional and innate-like lymphocytes from various lymphoid compartments including the spleen, mesenteric lymph nodes, lamina propria, and intra-epithelial lymphocytes. Mice colonized with IBS-D microbiota had a strong upregulation of neural genes involved in secretomotor function (VIP, ChAT, CALB), visceral sensitivity (NR2D and GABA-B), innate immunity (CD11c, CCR2, GATA-3, and GPR44) and regulation of epithelial integrity and control of commensal microbiota (Trefoil factor 3 and Lysozyme) compared to mice colonized with healthy microbiota. Lamina propria macrophages levels were higher in IBS-D colonized mice compared to healthy controls. Multi-parameter flow cytometry revealed similar frequencies of conventional T cells (CD3+CD4+ and CD3+CD8+) or B cells (CD19+B220+) in the spleen, MLN, and intestinal compartments between mice with IBS-D microbiota vs. healthy controls. However, there was a higher relative frequency of TCRαβ-TCRγδ -RORγt+ cells in the colonic lamina propria of mice with IBS-D microbiota compared to healthy controls. Our results demonstrate that the intestinal microbiota from patients with IBS-D and co-morbid anxiety alters multiple immune and neural system pathways involved in the regulation of gut function. IBS-D microbiota appears to affect the innate but not the adaptive immune system, with macrophages and innate lymphoid cells playing a key role. CIHROntario Graduate Scholarship (OGS) - Masters