Triptolide has been indicated potent anti-cancer effect involving multiple molecular targets and signaling pathways. High-mobility group box 1 (HMGB1) is a highly conserved DNA-binding protein taking part in breast cancer development. The therapeutic effect of triptolide on HMGB1 has not been reported. Thus, our study aims to clarify the role of HMGB1 in triptolide-induced anti-growth effect on breast cancer in vitro and in vivo. We demonstrated that triptolide significantly suppressed growth of breast cancer cells by inhibition of cell viability, clonogenic ability. Further studies evidenced that triptolide treatment not only inhibited HMGB1 mRNA expression, but also decreased supernatant level of HMGB1 in vitro. In line with these observations, exogenous recombinant HMGB1 promoted cell proliferation of breast cancer, and triptolide reversed the rHMGB1-promoted proliferative effect. As well, triptolide enhanced the anti-proliferative activity of EP (HMGB1 inhibitor). Furthermore, downstream correlation factors (TLR4 and phosphorylated-NF-κB p65) of HMGB1 were significantly decreased in vitro after triptolide treatment. Consistantly, we confirmed that tumor growth was significantly inhibited after triptolide treatment In vivo. Meanwhile, immunohistochemical analyses showed that triptolide treatment significantly decreased the level of cytoplasmic HMGB1 and TLR4 expression , whereas the expression of NF-κB p65 was relatively higher in cytoplasm, and conversely lower in nucleus as compared to the control group. Collectively, these results demonstrate that triptolide suppresses the growth of breast cancer cells via reduction of HMGB1 expression in vitro and in vivo, which may provide new insights into the treament of breast cancer.