Multiple genetic and signaling pathway alterations underlie the development of colon cancer. We utilized genome-wide transcriptome analysis to identify important gene expression patterns following treatment with 3,3'-diindolylmethane (DIM), a natural compound derived from cruciferous vegetables, on colon cancer cells. Statistical analyses of gene expression data from DIM treated cells revealed that 692 genes were significantly upregulated, while 731 genes were down-regulated. Putative gene networks showed that several oncogenes (β-catenin, Myc and FOS) were significantly suppressed by DIM treatment. Using clinical data from colon cancer patients, activation of β-catenin was found to be significantly associated with patient prognosis by Kaplan-Meir plot analysis. We validated the mRNA and protein expression levels of c-Myc, β-catenin, and cyclin D1, all of which were significantly suppressed after DIM treatment in DLD-1 and HCT116 cells. System level characterization of our findings suggests for the first time that β-catenin and c-Myc, which are major genes involved in colon carcinogenesis, were significantly downregulated by DIM treatment in colon cancer cells. Therefore, targeting Wnt/β-catenin signaling by DIM may be an attractive strategy for the prevention and treatment of colon cancer.