Methyl 2-trifluoromethyl-3,11-dioxo-18β-olean-1,12-dien-3-oate (CF3DODA-Me) is derived synthetically from glycyrrhetinic acid, a major component of licorice, and this compound induced reactive oxygen species (ROS) in RD and Rh30 rhabdomyosarcoma (RMS) cells. CF3DODA-Me also inhibited growth and invasion and induced apoptosis in RMS cells, and these responses were attenuated after cotreatment with the antioxidant glutathione, demonstrating the effective anticancer activity of ROS in RMS. CF3DODA-Me also downregulated expression of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and prooncogenic Sp-regulated genes including PAX3-FOXO1 (in Rh30 cells). The mechanism of CF3DODA-Me-induced Sp-downregulation involved ROS-dependent repression of c-Myc and cMyc-regulated miR-27a and miR-17/20a, and this resulted in induction of the miRNA-regulated Sp repressors ZBTB4, ZBTB10, and ZBTB34. The cell and tumor growth effects of CF3DODA-Me further emphasize the sensitivity of RMS cells to ROS inducers and their potential clinical applications for treating this deadly disease. IMPLICATIONS: CF3DODA-Me and HDAC inhibitors that induce ROS-dependent Sp downregulation could be developed for clinical applications in treating rhabdomyosarcoma.