MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate protein expression by cleaving or repressing the translation of target mRNAs. In mammals, their function mainly represses the target mRNA transcripts via imperfect complementary sequences in the 3'UTR of target mRNAs. Several miRNAs have been recently reported to be involved in modulation of glioma development, especially some upregulated miRNAs, such as microRNA-21 (miR-21), which has been found to function as an oncogene in cultured glioblastoma multiforme cells. Temozolomide (TMZ), an alkylating agent, is a promising chemotherapeutic agent for treating glioblastoma. Although chemotherapy with temozolomide may contain tumor growth for some months, invariable tumor recurrence suggests that cancer stem cells maintaining these tumors persist. Previous research showed that TMZ could inhibit the proliferation of human glioblastoma stem cells (GSC), but not induced apoptosis, which could supply the chance for glioblastoma recurrence. Accumulating evidence indicated that downregulation of miR-21 in glioblastoma cells caused repression of growth and increased apoptosis, all of which could theoretically enhance the chemotherapeutic effects of cancer therapy. In this study, we aimed to explore whether miR-21 downregulation could enhance the chemotherapeutic effects of TMZ and induce apoptosis on GSC. Interestingly, the results demonstrated that either miR-21 inhibitor or TMZ could not induce apoptosis on GSC. However, miR-21 inhibitor combined with TMZ significantly enhanced GSC apoptosis. Taken together, a combination of miR-21 inhibitor and TMZ could be an effective therapeutic strategy for GSC apoptosis to prevent potential glioblastoma recurrence.