Ischemia-reperfusion (IR) injury represents a major clinical challenge, which contributes to morbidity and mortality during surgery. The critical role of natural immunoglobulin M (IgM) and complement in tissue injury has been demonstrated. However, cellular mechanisms that result in the deposition of natural IgM and the activation of complement are still unclear. In this report, using a murine intestinal IR injury model, we demonstrated that the beta-actin protein in the small intestine was cleaved and actin filaments in the columnar epithelial cells were aggregated after a transient disruption during 30 min of ischemia. Ischemia also led to deposition of natural IgM and complement 3 (C3). A low dose of cytochalasin D, a depolymerization reagent of the actin cytoskeleton, attenuated this deposition and also attenuated intestinal tissue injury in a dose-dependent manner. In contrast, high doses of cytochalasin D failed to worsen the injury. These data indicate that ischemia-mediated aggregation of the actin cytoskeleton, rather than its disruption, results directly in the deposition of natural IgM and C3. We conclude that ischemia-mediated aggregation of the actin cytoskeleton leads to the deposition of natural IgM and the activation of complement, as well as tissue injury.