Background: This study aimed to investigate the inhibitory effect of imidazole on colon cancer cell proliferation and under- stand the mechanism involved. Material/Methods: MTT assay and flow cytometry using Hoechst 33258 staining were used to assess cell proliferation and morphology, respectively. Changes in protein expression was determined by western blotting assay. The reactive oxygen species (ROS) production in DLD-1 cells was analyzed by flow cytometry using DCFH-DA (2',7'-dichlorofluorescein diacetate) stain. Results: DLD-1 and HCT-116 cell viability was suppressed by imidazole in a concentration-based manner. At the concentration of 36 pM, imidazole reduced DLD-1 and HCT-116 cell viability to 22% and 28%, respectively. Treatment with imidazole led to chromatin material condensation, detaching of cells, and apoptotic nuclei. In imidazole treated cells, the G1/G0 phase cell proportion increased, whereas in the S and G2/M phases the cell proportion decreased. Imidazole treatment of DLD-1 cells markedly promoted activation of caspase-3, caspase-8, and caspase-9. The level of cleaved PARP1 was also upregulated in DLD-1 cells with imidazole treatment. Treatment of DLD-1 cells with imidazole suppressed BcI-2 and promoted Bax, p53, and cytc expression. The Akt activation was suppressed by imidazole treatment in DLD-1 cells. ROS generation in DLD-1 cells was enhanced markedly by treatment with imidazole. Conclusions: The present study demonstrated that imidazole inhibited colon cancer cell viability through activation of apoptosis and cell cycle arrest by increasing the generation of ROS, caspase activation, and apoptotic protein expression. Therefore, imidazole can act as a therapeutic molecule for the treatment of colon cancer.