Abstract Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B–deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia.