Keywords: caspase-3; colon carcinoma; monolayer culture; secoisolariciresinol diglucoside; spheroid culture; SW480 Abstract Apoptotic effects of secoisolariciresinol diglucoside (SDG) in 2D and 3D cultures of SW480 cells were investigated. 40-200 [MU]M SDG was used and IC50 values were determined for three different time intervals as 24, 48, or 72 hr for further experiments. BrdU, TUNEL, AIF, and caspase-3 stainings were used. SDG inhibited cell proliferation almost half and half for all time intervals in 2D and 3D cultures and also, induced apoptosis. Apoptotic cell percentages in the control group for 24, 48, and 72 hr were 27.00%, 29.00%, and 28.00%, respectively, while in the SDG treatment group were 59.00%, 61.00%, and 62.00%, respectively. In the spheroid cell culture, apoptotic cell percentages in the control group for 24, 48, and 72 hr were 6.90%, 7.20%, and 7.10%, respectively, while in the SDG treatment group were 19.50%, 19.50%, and 20.70%, respectively. Caspase-3 and AIF antibodies were used to indicate caspase-dependent and -independent apoptotic pathways. Significant increases were seen in both AIF and caspase-3 stainings when compared to the control group but caspase-3 staining results were significantly greater when compared to the AIF staining at all time intervals (p Practical applications Colon cancer is one of the most prevalent cancer in the developed countries and its etiology is complex. Although the underlying mechanisms are mostly unknown, the link between diet and colon cancer is known and dietary habits can promote cancer or protect against it. In recent years, flaxseed is accepted as a significant functional food ingredient and feeding with it could help in to prevent cancer. Secoisolariciresinol diglucoside is a flaxseed lignan and is metabolized to mammalian lignans by the gut. In the present study, SDG was evaluated for its apoptotic effects in colon carcinoma cell line via monolayer and spheroid cultures using immunohistochemical and gene expression techniques. Findings of this study suggest that SDG may protect against cancers and in particularly against colon cancer and further investigations has to be carried out for detailed underlying mechanisms.