Low and high molecular weight fucoidans (F_5-30K and F_>30K) were chemically modified through the addition of sulfate groups, and the effect of oversulfation on the in vitro anticancer activity was investigated. After the addition of sulfate groups, a considerable increase of 35.5 to 56.8% was observed in the sulfate content of the F_5-30K fraction, while the sulfate content of the F_>30K fraction increased to a lesser extent (from 31.7 to 41.2%). Significant differences in anticancer activity were observed between the oversulfated F_5–30K and F_>30K fractions, with activities of 37.3–68.0% and 20.6–35.8%, respectively. This variation in the anticancer activity of oversulfated fucoidan derivatives was likely due to differences in their sulfate content. The results suggest that the molecular conformation of these molecules is closely related to the extent of sulfation in the fucan backbones and that the sulfates are preferably substituted when the fucoidan polymers are in a loose molecular conformation.