The development of natural-based anti-tumor medicaments has acquired a great interest especially in the last few decades. Hence, cytotoxic activity of different fractions of fucoidan was evaluated. The fractions, produced from the total crude extract of the brown alga Fucus vesiculosus and purified by the recently-developed immobilized cationic dyes at different conditions, had different physicochemical properties and named fucoidan_1, fucoidan_6 and fucoidan_PDD. The activity of these fractions was studied in vitro against different kinds of cancerous mammalian cell lines including MCF-7 and Caco-2 and compared to their effects against skin primary fibroblasts. The results indicated a potent cytotoxic activity with regard to MCF-7 cells, while negligible (> 1500 mu g mL(-1)) towards primary fibroblasts. Moreover, higher general toxicity of crude fucoidan indicated that purification process succeeded to remove extraneous, co-extracted, cytotoxic compounds (e.g., polyphenols), which has a strong activity and possible interference in previously-published studies. Furthermore, a correlation was made between the cytotoxic activity and physico-chemical properties of fucoidan fractions, such as the sulfation degree and molecular weight. These findings reflected a real picture and expected low side effects regarding the cytotoxic activity of fucoidan purified by affinity chromatography.