Objective To investigate the effect of glycolysis inhibitor 2-DG on proliferation, migration and invasion of ErbB2 overexpressing breast cancer cell line MCF7/ ErbB2 and its molecular mechanism. Methods MCF7/ ErbB2 cells were treated with 2-DG (0, 0.5, 1, 2, 4, 8, 16, 32 mM) for 48 hours. Cell proliferation was detected by MTS assay. MCF7/ ErbB2 cells were treated with different concentration of 2-DG (0, 0.5, 1, 2 mM) for 24 hours. Cell migration and invasion was detected by Transwell assays. The glucose and lactate concentration in the culture media were determined by glucose and lactate assay kit, respectively, when MCF7/ ErbB2 cells were treated with 2-DG (0, 0.5, 1, 2 mM) for 48 hours, and then glucose uptake and lactate production by the cells were calculated. Finally, the cells were treated with 21 mM DG for different times (0, 4, 8, 12, 24 hours) to detect hexokinase II (HKII) protein levels by Western blot. Results Compared with untreated group, 2-DG ( 0.5-32 mM) significantly decreased the proliferation of MCF7/ ErbB2, 2-DG (0.5,1,2 mM) also inhibits the migration, invasion, glucose uptake and lactate production of MCF7/ ErbB2 cells (P＜0.001). Inhibition of cell proliferation, migration, invasion and glycolysis was 2-DG dose dependent. Moreover, 1 mM 2-DG downregulated HKII protein levels in MCF7/ ErbB2 cells significantly. Conclusion In breast cancer cell line MCF7/ ErbB2, 2-DG inhibits glycolysis by downregulating HKII protein levels leading to inhibition of proliferation, migration and invasion.