The Qing-Re-Yi-Liu decoction (QRYLD) is a clinically effective prescription for treatment of breast cancer. Our preliminary studies found that QRYLD treatment interfered with the Warburg effect in breast cancer cells. However, its chemical components and molecular mechanisms have not been clarified. This study analyzed the bioactive components of QRYLD aqueous extracts by HPLC. The effects of QRYLD on the malignant behaviors of MCF-7 cells and potential mechanisms were analyzed by CCK-8, transwell invasion, wound-healing assays, flow cytometry, Western blot assays and transcriptomic analysis as well as in vivo mouse tumor model. QRYLD aqueous extracts contained several bioactive components. Transcriptomic analysis indicated that QRYLD treatment altered the expression of several genes involved in biological processes and signaling pathways, such as manganese superoxide dismutase (MnSOD). Functionally, QRYLD, similar to MnSOD silencing, inhibited the malignant behaviors of MCF-7 cells and enhanced their apoptosis, whereas MnSOD overexpression had the opposite effects. Moreover, QRYLD treatment inhibited the Warburg effect by limiting glucose uptake and lactic acid production, and decreasing the relative expression of glucose transporter-1, hypoxia-inducible factor 1α, c-Myc, hexokinase-2, phosphofructokinase 1, lactate dehydrogenase A, pyruvate kinase isozyme 2, MnSOD, calmodulin-dependent kinase II (CaMKII), and AMP-activated protein kinase (AMPK) in MCF-7 cells. Finally, treatment with QRYLD significantly inhibited the growth of xenografted MCF-7 tumors in mice and reduced the tumor expression of MnSOD, CaMKII, and AMPK. These data suggest that QRYLD may target MnSOD to attenuate the MnSOD/CaMKII/AMPK signaling, inhibiting the Warburg effect and malignant behaviors in breast cancer.