Opioids are used widely by clinicians due to their potent analgesic activities and sedative properties. However, opioid use or abuse is associated with multiple adverse gastrointestinal (GI) symptoms and higher susceptibility to infection caused by pathogens with gut origin. Both clinical and laboratory studies implied that opioids showed suppressive effects on gut immunity and predisposed critically sick patients to infections while the mechanism underlying this defect is still unknown. In the present study we investigated how opioids modulate gut epithelial barrier function and immune responses of gut associated lymphoid tissue (GALT). We demonstrated significant bacterial translocation from gut lumen to mesenteric lymph node (MLN) and liver following morphine treatment in wild-type (WT) animals that was significantly attenuated in Toll-like receptor (TLR2 and 4) knockout mice. We further observed significant disruption of tight junction protein organization only in the ileum but not in the colon of morphine treated WT animals. Inhibition of myosin light chain kinase (MLCK) blocked the effects of both morphine and TLR ligands, suggesting the role of MLCK in tight junction modulation by TLR. Additionally we determined the immune responses of GALT to polymicrobial sepsis in the presence and absence of opioids by using a murine cecal ligation and puncture model. The results showed that opioids accelerated the mortality rate of polymicrobial sepsis. During sepsis progression, morphine treatment altered gut microbiome and subsequently promoted gram-positive bacterial dissemination, which induced excess IL-17A production in a TLR2-dependent manner, resulting in increased gut permeability, sustained inflammation and higher mortality. This study improved our understanding of the role of morphine in modulating gut barrier functions and the roles of GALT in infection susceptibility, which may provide the potential therapeutic targets for novel drug development and lead to more powerful strategy to control or prevent severe infectious diseases like sepsis especially in the opioid using and abusing population.