Previous studies indicate that endotoxin preconditioning may decrease the inflammatory response and alleviate intestinal mucosal damage caused by sepsis. However, it is not known whether preconditioning with endotoxin might protect the intestinal mucosa after hemorrhagic shock. In this study, we investigated the effect of lipopolysaccharide (LPS) preconditioning on the intestinal mucosa following hemorrhagic shock in a rat model. Given that intestinal toll-like receptor 4 (TLR4) signaling is exaggerated in response to LPS, we further investigated the role of TLR4 signaling in endotoxin tolerance. Animals were pre-treated with intra-peritoneal Escherichia coli LPS for 5 days prior to hemorrhagic shock. Animals were bled to achieve a mean arterial pressure (MAP) of 35-40 mmHg, then resuscitated with Ringer solution and the heparinized shed blood to maintain MAP between 90 and 100 mmHg. The distal ileum was harvested after resuscitation and graded for mucosal damage. TNF-alpha, TLR4, cleaved caspase-3, and intestinal trefoil factor 3 (TFF3) levels were measured at different time points. Pretreatment with LPS significantly reduced intestinal mucosal damage and protein levels of cleaved caspase-3. Furthermore, animals pre-treated with LPS experienced reduction of TNF-alpha and increased mucosal expression of TFF3. LPS tolerance was associated with reduced TLR4 expression. Endotoxin preconditioning can lessen the effects of ischemia and reperfusion injury in intestinal mucosa of a rat model with hemorrhagic shock. It is hypothesized that this effect is mediated via inhibition of TLR4 over-expression.