A growing number of studies have recently revealed a potential role for neutrophil extracellular traps (NETs) in the development of inflammation, coagulation and cell death. Deleterious consequences of NETs have been identified in ischemia-reperfusion (I/R)-induced organ damage, thrombosis and sepsis. And exogenous DNase-I has been suggested as a therapeutic strategy to attenuate ischemia-reperfusion (I/R) injuries in the kidney, brain and myocardium. Herein, we designed a study to investigate whether NETs contribute to the pathogenesis of intestinal I/R injury and evaluated the therapeutic value of DNase-1 in a rat model of intestinal I/R injury. In this rat model of intestinal I/R injury, we found that extracellular DNA was readily detectable in rat serum after 1 h of ischemia and 2 h of reperfusion. Treatment with DNase-1 significantly reduced the inflammatory response, restored intestinal barrier integrity and increased the expression of tight junction proteins. Our results indicate the existence of NETs in I/R-challenged intestinal tissues and firstly provide more evidence that DNase-1 may be an effective treatment for attenuating intestinal I/R injury.