Heat stroke (HS) is considered to be a severe systemic inflammatory reaction disease that is caused by high fever. The mortality of HS is high worldwide due to the lack of effective treatments. Presently, mesenchymal stem cells (MSCs) have been demonstrated to serve roles in inflammation and immune regulation. Therefore, the current study aimed to investigate the protective effect and mechanism of MSCs against the HS-induced inflammatory response and organ dysfunction. A rat model of HS was induced by a high-temperature environment and treated with MSCs via tail veins. The levels of molecular markers of organ function, inflammatory factors and chemokines were examined at days 1, 7, 14 and 28. Histological staining was performed on the intestines of rats and control groups, and the Chiu's scores of the two groups were compared. The results revealed that MSCs injection significantly reduced the mortality and inhibited the circulatory inflammatory response. Additionally, main organ function, such as in the liver and kidney, were significantly improved following MSCs infusion in HS rats. Furthermore, MSCs treatment significantly improved edema, necrosis and villus exfoliation of intestinal mucosa, and reduced the inflammatory response of intestinal tissue. These results indicated that MSC infusion had therapeutic effects on HS of rats by regulating the circulatory and intestinal inflammatory response. Moreover, MSCs may be able to protect organ function and promote tissue repair in HS. The results of the current study indicated that MSCs may be used as a potential method to treat HS and the resulting organ dysfunction.