Loss of the intestinal barrier is critical to the clinical course of heat illness, but the underlying mechanisms are still poorly understood. We tested the hypothesis that conditions characteristic of mild heatstroke in mice are associated with injury to the epithelial lining of the intestinal tract and comprise a critical component of barrier dysfunction. Anesthetized mice were gavaged with 4 kDa FITC-dextran (FD-4) and exposed to increasing core temperatures, briefly reaching 42.4°C, followed by 30 min recovery. Arterial samples were collected to measure FD-4 concentration in plasma (in vivo gastrointestinal permeability). The small intestines were then removed to measure histological evidence of injury. Hyperthermia resulted in a ≈2.5-fold elevation in plasma FD-4 and was always associated with significant histological evidence of injury to the epithelial lining compared with matched controls, particularly in the duodenum. When isolated intestinal segments from control animals were exposed to ≥41.5°C, marked increases in permeability were observed within 60 min. These changes were associated with release of lactate dehydrogenase, evidence of protein oxidation via carbonyl formation and histological damage. Coincubation with N-acetylcysteine protected in vitro permeability during hyperthermia and reduced histological damage and protein oxidation. Chelation of intracellular Ca(2+) to block tight junction opening during 41.5°C exposure failed to reduce the permeability of in vitro segments. The results demonstrate that hyperthermia exposure in mouse intestine, at temperatures at or below those necessary to induce mild heatstroke, cause rapid and substantial injury to the intestinal lining that may be attributed, in part, to oxidative stress.