The nucleation, growth and aggregation of calcium oxalate (CaOx) crystals and the oxidative damage of renal tubular epithelial cells are the key factors to induce kidney stones. In this study, degraded Porphyra yezoensis polysaccharide (PYP0) with 14.14% sulfate group (–OSO3−) content was modified via the sulfur trioxide–pyridine method to obtain three kinds of sulfated P. yezoensis polysaccharides (PYPs), namely, PYPS1, PYPS2, and PYPS3, with –OSO3− group contents of 17.11%, 20.28%, and 27.14% respectively. Fourier transform infrared spectroscopy, 1H NMR, and 13C NMR analyses showed that the –OSO3− groups replaced the hydroxyl groups at the C2, C4, and C6 positions on (1 → 3)-linked β-D-galactose, the basic structural skeleton unit of PYP0. The antioxidant activity of the PYPSs increased after sulfation, and their scavenging capacity for OH and DPPH free radicals was enhanced with the increase in their –OSO3− group content. Calcium oxalate (CaOx) crystal growth experiments showed that sulfated PYPs promoted the conversion of the thermodynamically stable and sharp CaOx monohydrate (COM) crystals into the thermodynamically unstable and round CaOx dihydrate crystals. With the increase in the –OSO3− group content of the polysaccharides, the concentration of soluble Ca2+ ions in the supernatant increased and the amount of CaOx precipitate decreased. PYPs were nontoxic to human kidney proximal tubular epithelial cells (HK-2) and could protect HK-2 from oxidative damage caused by nano-COM and reduce the level of reactive oxygen species in cells. PYPS3, which had the highest degree of sulfation, had the best protective capability. The results of this work showed that sulfation improved the biological activity of PYPs. This study could provide inspiration for the development of new drugs for the prevention and treatment of kidney stones.