The mammalian intestine contains a dense and diverse community of microorganisms. The resident microbiota makes contributions to host to promote proper immune system development and limit pathogen colonization. In this study, the effects of microbiota disruption with or without TLRs stimulation on intestinal permeability and immunity were examined in C57BL/6 mice receiving antibiotic treatment for 6 days and in antibiotics-treated mice received dead E. coli or S. aureus at day 4. The results showed that antibiotic treatment significantly decreased the total number of bacteria including specific aerobic group Enterobacteriaceae and Enterococcus, and specific anaerobic group Lactococcus/Bifidobacterium in intestinal mucosa and lumen. Although only a slight increase in the intestinal permeability and no change in caspase-3 activity of intestinal mucosa were observed after antibiotic treatment, the bacterial translocation (BT) to mesenteric lymph nodes (MLN) increased significantly. Subsequent experiments showed that antibiotic treatment decreased the mucosal killing activity and the expression of non-defensin family including RegIII£], RegIII£^, CRP-ductin and RELM£] but not the defensin family, and increased the translocation of pathogen K. pneumoniae significantly, suggesting that the increase of BT to MLN after antibiotic treatment is likely due to a reduction in gut immunity rather than an increase of intestinal permeability. Moreover, stimulation of TLR4 reversed the effect of antibiotic treatment, suggesting that the functioning of TLR4 in intestinal epithelium is required to prevent pathogenic invasion and maintain intestinal homeostasis.