The present study assessed the effect of the lipid metabolism, fat mass and the obesity-associated gene (FTO), on energy metabolism of breast cancer cells. The human breast cancer cell lines, MCF-7 and MDA-MB-231, and HCC1937 human breast cells were studied. Real-time PCR was used to measure the levels of FTO mRNA from breast cancer cells and normal breast cells. MDA-MB-231 cells were transfected with miFTO inhibitor or inhibitor control, and cells were assessed for levels of lactic acid, ATP, pyruvate kinase activity, and hexokinase activity assay using specific kits. Western blot analysis was used to measure the levels of phosphatidylinositol 3-kinase (PI3K), p-PI3K, protein kinase B (Akt) and p-Akt in transfected breast cancer cells. The expression of FTO was significantly increased in MCF-7 and MDA-MB-231 cells compared with HCC1937 cells (P<0.01). The lactic acid content of breast cancer cells transfected with the miFTO inhibitor was significantly lower compared with cells transfected with the miFTO inhibitor control and nontransfected cells (P<0.05). The ATP content of breast cancer cells transfected with the miFTO inhibitor was significantly lower compared with the control group and inhibitor control group (P<0.05). The pyruvate kinase activity and hexokinase activity of breast cancer cells transfected with the miFTO inhibitor were significantly lower compared with the control group and inhibitor control group (P<0.01). Western blot analysis showed that after breast cancer cells were transfected with the miFTO inhibitor, the levels of PI3K, p-PI3K, Akt and p-Akt were significantly lower than in the control group and inhibitor control group. In conclusion, the FTO gene is overexpressed in breast cancer cells. Overexpression of the FTO gene can promote breast cancer cell glycolysis and the mechanism is related to the PI3K/AKT signaling pathway.