Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Patients diagnosed with GBM have a poor prognosis, and it has been reported that tumor malignancy and GBM recurrence are promoted by STAT3 signaling. As resveratrol (RV), a polyphenol in grapes, is reported to be a potent and non‐toxic cancer‐preventive compound, the aim of this study was to investigate the therapeutic effect and molecular mechanisms of RV on GBM‐derived radioresistant tumor initiating cells (TIC). Firstly, our results showed that primary GBM‐CD133⁺ TIC presented high tumorigenic and radiochemoresistant properties as well as increased protein levels of phosphorylated STAT3. We consistently observed that treatment with shRNA‐STAT3 (sh‐STAT3) or AG490, a STAT3 inhibitor, significantly inhibited the cancer stem‐like cell properties and radioresistance of GBM‐CD133⁺ in vitro and in vivo. Furthermore, treatment of GBM‐CD133⁺ with 100 µM RV induced apoptosis and enhanced radiosensitivity by suppressing STAT3 signaling. Microarray results suggested that RV or AG490 inhibited the stemness gene signatures of GBM‐CD133⁺ and facilitated the differentiation of GBM‐CD133⁺ into GBM‐CD133⁻ or astrocytoma cells. Finally, xenotransplant experiments indicated that RV or sh‐STAT3 therapy could significantly improve the survival rate and synergistically enhance the radiosensitivity of radiation‐treated GBM‐TIC. In summary, RV can reduce in vivo tumorigenicity and enhance the sensitivity of GBM‐TIC to radiotherapies through the STAT3 pathway. J. Cell. Physiol. 227: 976–993, 2012. © 2011 Wiley Periodicals, Inc.