Previous studies from our laboratory have shown chemopreventive effects of dietary flaxseed on azoxymethane-induced colon tumor development in male Fischer rats and Apc(Min) mice. Tumorigenesis is associated with uncontrolled cell growth and loss of apoptosis. Accordingly, the objective of this investigation was to study the effects of mammalian lignans (enterodiol and enterolactone) and ω-3 polyunsaturated fatty acid α-linolenic acid, principal active components in flaxseed on cell proliferation and apoptosis in human colon adenocarcinoma Caco-2 cells, thus elucidating possible mechanism of action. BrdU incorporation assay was used for cell proliferation and fluorescence-activated cell sorting (FACS) analysis of annexin V/propidium iodide staining was used for determining apoptotic cells. Results showed that enterodiol, enterolactone and α-linolenic acid at different concentrations caused a significant (p < 0.05) increase in apoptotic cells and decrease in cell proliferation. Therefore, dietary flaxseed containing α-linolenic acid and lignans causes a decrease in cell proliferation and an increase in apoptosis resulting in the effective chemoprevention for intestinal and colon tumor development.