Polysaccharides (PS) are important bioactive components in medicinal mushrooms. However, sulfated polysaccharides (SPS), which are characterized by the presence of sulfate groups on monosaccharides and known for their remarkable bioactivities, have never been reported in mushroom fruiting bodies. Our previous study found that Laetiporus sulphureus, an edible and medicinal mushroom in Taiwan, contained a high amount of SPS (Lu et al., Int. J. Biol. Macromol. 2023, 226:1236-1247). This study aimed to investigate the differences in physicochemical properties between PS and SPS of L. sulphureus fruiting bodies and evaluate their anti-breast cancer activity using a triple-negative breast cancer cell line (MDA-MB-231). Results showed that the physicochemical properties of PS and SPS were significantly different, including the contents of sulfate (0.16% vs. 16.17%), protein (0.43% vs. 4.33%), and uronic acid (10.98% vs. 3.43%), as well as the molecular weight, the monosaccharide composition, and the conformation. SPS exhibited more potent anti-proliferative activity against MDA-MB-231 cells than PS. Furthermore, it had a selective cytotoxic effect on breast cancer cells but not the normal breast epithelial cells (H184B5F5/M10). SPS induced G0/G1 cell cycle arrest via down-regulating CDK4 and cyclin D1 protein expression, and up-regulating p21 protein expression. It also markedly inhibited breast cancer cell migration at low concentrations. In conclusion, SPS, which had higher sulfate and protein contents and diversified monosaccharide composition, exhibited more potent than PS in inhibiting MDA-MB-231 cell proliferation. SPS could induce cell cycle arrest at G0/G1 phase and inhibit breast cancer cell migration.