Pyropia yezoensis Sookwawon 104 is a newly cultivated strain of red marine algae. The present study aimed to investigate the in vitro antiproliferative activity of sulfated polysaccharide extracted from P. yezoensis Sookwawon 104 (PYSP), as well as that of its low molecular weight (Mw) derivatives. PYSP is a heterogeneous sulfated polysaccharide mainly composed of galactose, glucose and fucose. PYSP was degraded by gamma-irradiation at doses of 20 and 100 kGy to produce two derivatives, named as PYSP-20 and PYSP-100, respectively. Comparison of PYSP, PYSP-20 and PYSP-100 revealed clear differences in their molecular weight (Mw) distributions, and distinct in vitro antiproliferative activities against Hep3B, MDA-MB-231 and HeLa cancer cell lines. PYSP-20 and PYSP-100 exhibited stronger antiproliferative effects than PYSP, suggesting that the reduction in Mw may have increased the in vitro antiproliferative activity. Furthermore, the mRNA expression levels of the antitumor gene P53 and cell cycle-associated genes P21, Cyclin B1 and cyclin dependent kinase 1 (Cdk1) were further analyzed by reverse transcription-quantitative PCR in PYSP-20 and PYSP-100-treated cancer cells. PYSP and its derivatives were shown to inhibit the proliferation of tumor cells by regulating the expression of P53, P21, Cyclin B1 and Cdk1. In conclusion, low-Mw polysaccharide derivatives prepared from P. yezoensis Sookwawon 104 by gamma-irradiation exhibit significant inhibition effects on cancer cell proliferation in vitro and may be a novel source of potential anticancer therapeutic agents.