P-glycoprotein (P-gp) protects the mammalian intestinal epithelium by effluxing toxins from the epithelial cells as well as release of human endocannabinoids that inhibit neutrophil infiltration. Diminished or dysfunctional P-gp is associated with intestinal inflammation including ulcerative colitis (UC). Due to the microbiome dysbiosis associated with UC, we hypothesize that the healthy microbiota promote colonic P-gp expression. Utilizing mouse models of antibiotic treatment, microbiota reconstitution, and metabolite perturbation, we have shown butyrate and secondary bile acids, dependent on vancomycin-sensitive bacteria, induce P-gp expression in vivo. We have shown these metabolites together potentiate induction of P-gp in intestinal epithelial cell lines in vitro, which is sufficient to inhibit primary human neutrophil transmigration. Furthermore, in UC patients we find diminished P-gp expression is coupled to reduction of anti-inflammatory endocannabinoids and luminal content with reduced capability to induce P-gp expression. Additionally, we have found butyrate contributes to P-gp expression via histone deacetylase inhibition, and secondary bile acids regulate P-gp expression via nuclear receptors pregnane X receptor and vitamin D receptor. Employing RNA sequencing (RNAseq) in IECs uncovered signaling networks that are uniquely triggered with the combination of butyrate and secondary bile acids, suggesting additional pathways required for maximal P-gp expression in the colon. Together we identify a mechanistic link between cooperative functional outputs of the complex microbial community and suppression of intestinal inflammation. These data emphasize the importance of the intestinal microbiome in driving the P-gp axis to suppress aberrant neutrophil infiltration and identify potential therapeutic targets for promoting P-gp expression in an inflamed colon to reset homeostasis.