Hyperthermia, the procedure of raising the temperature of tumor-loaded tissue to 40 degrees -43 degrees C, has been applied to various established cancer treatments. Although the mechanism of hyperthermia in cancer treatment is well-known, there are few or no studies regarding the effect of hyperthermia on the tumor-supportive stroma. Mesenchymal stem cells (MSCs) display the potential for differentiation into various tissues. MSCs are also reported to play a role as potential precursors for tumor stroma in providing a favorable environment for tumor progression. Here, we investigated the effects of hyperthermia-treated MSCs on the viability and growth of cancer cells. Culture supernatants from non-shocked or heat-shocked MSCs (NS-MSCs or HS-MSCs) were added to MCF7 cells. Morphological analysis and cell proliferation assay showed the reduced viability and growth of MCF7 cells by addition of culture medium conditioned by HS-MSCs. Additionally, exposure to the conditioned medium by HS-MSCs induced cell cycle arrest at G2/M phase, increased MHC class I, Fas receptor, and TNF-R expressions, and decreased MDR1 expression in the MCF7 cells. In particular, the conditioned medium of HS-MSCs accelerated the inhibition of tumor cell growth by several chemotherapeutic drugs. These data present new aspects of hyperthermia in cancer treatment, suggesting that hyperthermia can enable tumor stroma provide a sensitizing environment for tumor cells to undergo cell death.