ErbB2 has been shown to activate signaling molecules that may regulate glucose metabolism. However, there is no evidence reported to directly link ErbB2 to glycolysis, and the mechanism underlying ErbB2-enhanced glycolysis is poorly understood. In this study, we investigated the role and mechanism of ErbB2 in regulating glycolysis. We found that ErbB2-overexpressing cells possessed a significantly higher level of glycolysis when compared to the ErbB2-low-expressing cells, and the downregulation of ErbB2 markedly decreased glycolysis. Overexpression of ErbB2 increased the expression of glycolysis-regulating molecules lactate dehydrogenase A (LDH-A) and heat shock factor 1 (HSF1). ErbB2 activated HSF1, indicated by the increased HSF1 trimer formation, and promoted HSF1 protein synthesis. HSF1 bound to LDH-A promoter and the downregulation of HSF1 reduced the expression of LDH-A and subsequently decreased cancer cell glycolysis and growth. Moreover, the glycolysis inhibitors, 2-deoxyglucose and oxamate, selectively inhibited the growth of ErbB2-overexpressing cells. Taken together, this study shows that in human breast cancer cells, ErbB2 promotes glycolysis at least partially through the HSF1-mediated upregulation of LDH-A. This pathway may have a major role in regulating glucose metabolism in breast cancer cells. These novel findings have important implications for the design of new approaches to target ErbB2-overexpressing breast cancers. © 2009 Macmillan Publishers Limited All rights reserved.