ABSTRACT Acute mesenteric ischemia is associated with high morbidity and mortality. In recent studies, we found that the intestine is an important source of matrix metalloproteinase (MMP)8 during intestinal injury. We hypothesized that genetic ablation or pharmacological inhibition of MMP8 would reduce intestinal injury in mice subjected to intestinal ischemia–reperfusion (I/R) injury. Male mice aged 8–12 wk were subjected to intestinal I/R injury by transient occlusion of the superior mesenteric artery for 30 min. MMP8 was inhibited by genetic and pharmacological approaches. In vivo10th and 90th percentiles study endpoints included several functional, histological, and biochemical assays. Intestinal sections were assessed for barrier function and expression of tight junction proteins. I/R injury led to increased intestinal and systemic expression of MMP8. This increase was associated with increased intestinal neutrophil infiltration, epithelial injury, and permeability. I/R injury was associated with increased systemic inflammation and weight loss. These parameters were ameliorated by inhibiting MMP8. I/R injury caused a loss of the tight junction protein claudin‐3, which was ameliorated by genetic ablation of MMP8. MMP8 plays an important role in intestinal I/R injury through mechanisms involving increased inflammation and loss of claudin‐3. Inhibition of MMP8 is a potential therapeutic strategy in this setting.—Daly, M. C., Atkinson, S. J., Varisco, B.M., Klingbeil L., Hake, P., Lahni, P., Piraino, G., Wu, D., Hogan, S. P., Zingarelli, B., Wong, H.R. Role of matrix metalloproteinase‐8 as a mediator of injury in intestinal ischemia and reperfusion. FASEB J. 30, 3453–3460 (2016). www.fasebj.org