An increased risk of colorectal cancer is related to the development of metabolic syndromes including hyperglycemia, and hyperinsulinemia. The high circulatory levels of glucose and/or insulin or the application of exogenous insulin may promote carcinogenesis, cancer progression and metastasis, which can be attributed to the Warburg effect or aerobic glycolysis. We attempted to resolve these existing questions by applying the glucose analog 2-deoxyglucose (2DG). According to the in vitro studies we performed, the glycolysis of colorectal cancer cells could be interrupted by 2DG as it decreased the cellular productions of ATP and lactate. In addition, 2DG induced apoptosis and cell cycle arrest, and inhibited proliferation, migration and invasion of these cells. Since insulin can stimulate the cellular uptake of hexose, including 2DG, the combination of 2DG and insulin improved the cytotoxicity of 2DG and meanwhile overcame the cancer-promoting effects of insulin. This in vitro study provided a viewpoint of 2DG as a potential therapeutic agent against colorectal cancer, especially for patients with concomitant hyperinsulinemia or treated with exogenous insulin.