Background Heatstroke is associated with exposure to high ambient temperature (AT) and relative humidity (RH), and an increased risk of organ damage or death. Previously proposed animal models of heatstroke disregard the impact of RH. Therefore, we aimed to establish and validate an animal model of heatstroke considering RH. To validate our model, we also examined the effect of hydration and investigated gene expression of cotransporter proteins in the intestinal membranes after heat exposure. Methods Mildly dehydrated adult male C57/BL6J mice were subjected to three AT conditions (37 degrees C, 41 degrees C, or 43 degrees C) at RH > 99% and monitored with WetBulb globe temperature (WBGT) for 1 h. The survival rate, body weight, core body temperature, blood parameters, and histologically confirmed tissue damage were evaluated to establish a mouse heatstroke model. Then, the mice received no treatment, water, or oral rehydration solution (ORS) before and after heat exposure; subsequent organ damage was compared using our model. Thereafter, we investigated cotransporter protein gene expressions in the intestinal membranes of mice that received no treatment, water, or ORS. Results The survival rates of mice exposed to ATs of 37 degrees C, 41 degrees C, and 43 degrees C were 100%, 83.3%, and 0%, respectively. From this result, we excluded AT43. Mice in the AT 41 degrees C group appeared to be more dehydrated than those in the AT 37 degrees C group. WBGT in the AT 41 degrees C group was > 44 degrees C; core body temperature in this group reached 41.3 +/- 0.08 degrees C during heat exposure and decreased to 34.0 +/- 0.18 degrees C, returning to baseline after 8 h which showed a biphasic thermal dysregulation response. The AT 41 degrees C group presented with greater hepatic, renal, and musculoskeletal damage than did the other groups. The impact of ORS on recovery was greater than that of water or no treatment. The administration of ORS with heat exposure increased cotransporter gene expression in the intestines and reduced heatstroke-related damage. Conclusions We developed a novel mouse heatstroke model that considered AT and RH. We found that ORS administration improved inadequate circulation and reduced tissue injury by increasing cotransporter gene expression in the intestines.