Sulfated polysaccharides isolated from Enteromorpha prolifera and fractionated using ion exchange and gel permeation chromatography were investigated to determine their molecular characteristics and biological activities. The crude and fractionated polysaccharides ($F_1$, $F_2$, and $F_3$) consisted mostly of carbohydrates (50.8-62.5%), sulfates (14.5-18.8%), and uronic acid (13.8-16.6%) with different levels of monosaccharides, including rhamnose (57.1-87.6%), glucose (3.6-39.1%), and xylose (2.4-8.8%). The protein content ranged from 1.0 to 13.9 %, and after the fractionation, most proteins were included in the F2 fraction (11.3%). The crude, $F_1$, $F_2$, and $F_3$ exhibited the molecular weights (Mw) of $37-1,281{ imes}10^3;g/mol$. The polysaccharides had no significant direct cytotoxicity to cancer cells AGS or DLD-1. On the other hand, the $F_1$ and $F_2$ stimulated Raw 264.7 cells to produce a considerable amount of nitric oxide (NO). The investigation of $F_2$ subfractions failed to reveal a specific subfraction more significantly affecting the NO production, thus requiring further systematic elucidation of their structural characteristics.