There is growing appreciation for the importance of gastrointestinal microbiota in many physiological and pathophysiological processes. While morphine and other narcotics are the most widely prescribed therapy for moderate to severe pain clinically, they have been noted to alter microbial composition and promote bacterial translocation to other tissues. Here we examined the pharmacodynamic properties of chronic morphine in mice following bacterial depletion with oral gavage of an antibiotic cocktail (ABX). ABX significantly reduced gut bacteria and prevented chronic morphine induced increases in gut permeability, colonic mucosal destruction, and colonic IL-1β expression. In addition, ABX prevented the development of antinociceptive tolerance to chronic morphine in both the tail-immersion and acetic acid stretch assays. Morphine tolerance was also reduced by oral vancomycin that has 0% bioavailability. These findings were recapitulated in primary afferent neurons isolated from dorsal root ganglia (DRG) innervating the lower gastrointestinal tract, wherein in-vivo administration of ABX prevented tolerance to morphine-induced hypoexcitability. Finally, though ABX repeatedly demonstrated an ability to prevent tolerance, we show that it did not alter susceptibility to precipitation of withdrawal by naloxone. Collectively, these finding indicate that the gastrointestinal microbiome is an important modulator of physiological responses induced by chronic morphine administration.