Cancers are characterized by reprogrammed glucose metabolisms to fuel cell growth and proliferation. Carbohydrate response element binding protein (ChREBP) is a glucose-mediated transcription factor that strongly regulates glycolytic and lipogenic pathways. It has been shown to associate with metabolic diseases, such as obesity, diabetes and non-alcoholic fatty liver diseases. However, how it associates with cancers has not been well understood. In this study, ChREBP expression was assessed by immunohistochemistry in colon tissue arrays containing normal colon tissue and cancer tissue at different clinical stages. Tissue mRNA levels of ChREBP were also measured in a cohort of colon cancer patients. We found that ChREBP mRNA and protein expression were significantly increased in colon cancer tissue compared to healthy colon (p < 0.001), and their expression was positively correlated to colon malignancy (for mRNA, p = 0.002; for protein p < 0.001). Expression of lipogenic genes (ELOVL6 and SCD1) in colon cancer was also positively associated with colon malignancy (for both genes, p < 0.001). In vitro, ChREBP knockdown with siRNA transfection inhibited cell proliferation and induced cell cycle arrest without changes in apoptosis in colon cancer cell lines (HT29, DLD1 and SW480). Glycolytic and lipogenic pathways were inhibited but the p53 pathway was activated after ChREBP knockdown. Taken together, ChREBP expression is associated with colon malignancy and it might contribute to cell proliferation via promoting anabolic pathways and inhibiting p53. In addition, ChREBP might represent a novel clinical useful biomarker to evaluate the malignancy of colon cancer.