Previous studies have shown that hyperglycemia is connected to the malignant progression of breast cancer; however, the effects of hyperglycemia on tumorigenic potential in breast cancer cells are largely unknown. Here, we demonstrated that the ability of the human breast cancer cell line MCF-7 to undertake anchorage-independent colony growth was significantly enhanced when cultured under high-glucose conditions compared with that under physiological glucose conditions. The high-glucose conditions also promoted phosphorylation of Akt, suggesting that MCF-7 cells cultured in these conditions acquired an increased ability to undergo anchorage-independent growth at least in part through Akt activation, which has been linked to the development of breast cancer. These results raise the possibility that regulation of Akt activity contributes to the tumorigenesis of breast cancer under high-glucose conditions, and we propose that additional analyses of high glucose-induced tumor formation would provide novel strategies for the diagnosis and therapy of breast cancer with hyperglycemia.