Upregulation of glycolysis was often observed in human HER2-overexpressing cancers. In this study, we demonstrated that KU004, a dual novel EGFR/HER2 inhibitor, disrupted cancer cell proliferation via modulation of glycolysis. KU004, inhibited the Warburg effect by suppressing hexokinase II (HK2) expression at the transcriptional and post-translational levels. Further study demonstrated that the downregulation of HKII by KU004 was mainly mediated by the PI3K/Akt signaling pathway. Furthermore, the role of HKII downregulation in KU004-mediated antitumor effect was also confirmed in our in vivo xenograft model. Collectively, these data suggest that multifaceted targeting the aberrant glucose metabolism along with the upstream HER2 may be an effective approach for clinical treatment against HER2+ cancer.