Gut-derived endotoxin and pathogenic bacteria have been proposed to be important causative factors of morbidity and mortality due to heat stroke. However, the molecular mechanisms underlying the small intestinal lesions caused by heat stroke have yet to be well characterized. In order to verify the possible inflammatory pathogenesis of intestinal tissue injury, a mouse heat stroke model was established. Cooling treatment was applied, mimicking the clinical therapy. Morphologic changes in intestinal tissue and 10 cytokines and chemokines produced from ileum tissue were detected, and their correlation was analyzed. As a result, intestinal lesions in mice worsened with the increase in rectal core temperature (Tc) during heat stress. When heat stress was halted at a Tc of no more than 41°C followed by cooling treatment, pathological recovery from the injury of heat stress was observed. However, when cooling treatment was applied after the Tc reached 42°C, the lesions continuously deteriorated until the animals succumbed. The levels of pro-inflammatory (IL-1β, IL-2, IL-6 and TNF-α) and anti-inflammatory (IL-10, IL-12p40) cytokines showed significant changes at different time points during the heat stress and cooling treatment, but no changes in GM-CSF, MCP-1, MIP-1α and IL-4 were observed. Levels of IL-1β, IL-10 and IL-12p40 were moreover significantly correlated with intestinal injury scores. A significant inverse linear correlation was observed between intestinal injury and IL-12p40 levels. In conclusion, this study provided insight into the inflammatory pathogenesis of intestinal tissue injury after heat stress and cooling treatment in a mouse model, and presented a potential biomarker for the evaluation of intestinal injury during heat stroke.