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Background: Arsenic trioxide (As2O3) has been demonstrated to suppress tumorigenesis in human glioma. However, the exact molecular mechanisms by which As2O3 exerts its tumor suppressor functions are elusive. Therefore, it is warranted to explore the underlying mechanism of As2O3–mediated anti-tumor activity in glioma. Methods: To achieve our goal, we used multiple approaches including MTT assay, apoptosis, Real-time RT-PCR, Western blotting, invasion assay, and gene transfection. Results: We observed that A22O3 inhibited cell growth and induced apoptosis as well as suppressed migration and invasion in human glioma cells. Moreover, we found that As2O3 down-regulated miR-125b expression and subsequently up-regulated its target gene Bak1 expression. Furthermore, we identified that As2O3 exerts its anti-tumor activity partly through regulation of miR-125b. Conclusions: Our present study suggests that As2O3 could be a potential therapeutic agent for treatment of human glioma.