MicroRNAs (miRNAs) regulate cell proliferation and differentiation by affecting gene expression at the post-transcriptional level by binding to complementary sequences within mRNAs in cancer cells, indicating that miRNAs can function as tumor suppressors or oncogenes. Recent studies showed that dysregulation of miRNA expression was associated with increased tumorigenicity and poor prognosis in several types of cancers, including Ewing's sarcoma (ES). To explore possible oncogenic factors in ES, we conducted micro-array-based investigation and profiled the changes in miRNA expression and their effects on downstream mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs). miR-301a was significantly upregulated, while the phosphatase and tensin homolog (PTEN) expression was significantly downregulated in all tested ES cells as compared to hMSCs. When anti-miR-301a was transfected into ES cell lines, PTEN expression was significantly enhanced, suggesting that PTEN might be a target of miR-301a in ES cells. The expression of protein kinase B (Akt), which is inversely correlated with PTEN expression, was significantly downregulated in anti-miR-301a-transfected cells. Additionally, the transfection of anti-miR-301a inhibited ES cell proliferation and cell cycle progression. Furthermore, downregulation of miR-301a in ES cells significantly suppressed tumor growth in vivo. Our results demonstrated the novel mechanism controlling PTEN expression via miR-301a in ES cells. Given that PTEN is a pivotal phosphatase factor that regulates cell cycle progression, apoptosis, and proliferation, these results might lead to development of new ES-related therapeutic targets.