Opioids such as morphine have many beneficial properties as analgesics, however, opioids may induce multiple adverse gastrointestinal symptoms. It has been recently demonstrated that morphine treatment results in significant disruption in gut barrier function leading to increased translocation of gut commensal bacteria. However, it is unclear how opioids modulate gut homeostasis. A mouse model was used to investigate the effects of morphine treatment on gut microbiome and metabolome. When phylogenetic profiles of gut microbes were characterized, the results revealed a significant shift in the colonic microbiome following morphine treatment when compared to placebo. At the species level, Enterococcus faecalis was associated with morphine-modulated gut microbiome alteration. Morphine treatment also resulted in dramatic changes in the fecal metabolomic profile. Through LC-MS based metabolomics profiling analysis, fatty acids and bile acids metabolism and in particular, deoxycholic acid (DCA) and phosphatidylethanolamines (PEs) was identified to be greatly affected by morphine treatment, implicating that changes in the microbiome community has functional consequences. In a longitudinal study, naltrexone, an opioid receptor antagonist, reversed the effect of morphine on bile acid metabolism, indicating morphine induced changes are opioid receptor dependent. Cross-correlation between gut microbiome and metabolome indicated association between bacterial communities and functional metabolites. Furthermore, morphine induced dysbiosis disrupts morphine metabolism and its enterohepatic recirculation. This study shed light on the effects of morphine on the microbiome-metabolome-host axis, and its role in gut homeostasis. In a mouse model of Citrobacter rodentium infection, morphine treatment resulted in 1) the promotion of C. rodentium systemic dissemination, 2) increase in virulence factors expression with C. rodentium colonization in intestinal contents, 3) altered gut microbiome, 4) damaged integrity of gut epithelial barrier function, 5) inhibition of C. rodentium-induced increase of goblet cells, and 6) dysregulated IL-17A immune response. This is the first study to demonstrate that morphine promotes pathogen dissemination in the context of intestinal C. rodentium infection, indicating morphine modulates virulence factor-mediated adhesion of pathogenic bacteria and induces disruption of mucosal host defense during C. rodentium intestinal infection in mice.