To explore the effect and mechanisms of cytoplasmic M-CSF on glucose metabolism in human breast cancer MCF-7 cells, MCF-7 cells stable expressing cytoplasmic M-CSF were constructed. Relative ATP content was measured by ATP assay kit, glucose was measured using glucose assay kit and lactate was measured using lactate acid assay kit. The expression of HK2, PKM2 and GLUT-1 in three kinds of cell with LY294002 or API-2 was detected by West-blotting. The sensitivity of MCF-7 and MCF-7-M cells to 5-FU with the treatment of ATP depletion by 3-BrPA was observed by MTT assay. It was found that the ATP level of MCF-7-M cells was significantly higher than that of MCF-7 cells (P<0.05); 2-DG decreases the ATP level of MCF-7 and MCF-7-M cells, and the effect of lowering the ATP level of MCF-7-M cells is more obvious (P<0.01). The glucose uptake and lactate secretion of MCF-7-M cells were significantly higher than those of MCF-7 cells (P<0.01). After the treatment with API-2, the glucose consumption and lactate secretion of MCF-7 and MCF-7-M cells were significantly reduced (P<0.01). The expressions of GLUT-1, HK2 and PKM2 in MCF-7-M cells were significantly higher than those in MCF-7 cells (P<0.01). Both LY294002 and API-2 inhibited the expression of GLUT-1 in MCF-7-M cells (P<0.05). After the treatment with 3-BrPA, the drug sensitivity of MCF-7-M and MCF-7 cells to 5- FU was significantly enhanced (P<0.01). In conclusion, cytoplasmic M-CSF activates glycolysis by induce GLUT-1, HK2 and PKM2 protein expression in MCF-7 cells; PI3K/AKT signaling involves the pathway that glycolysis was activated by cytoplasmic M-CSF in MCF-7 cells.