Algal sulfated polysaccharides are known to be effective hyaluronidase inhibitors. We evaluated hyaluronidase inhibitory activity of sulfated polysaccharide (SP) from Caulerpa lentillifera. Results showed that SP with IC50 of 163 µg/mL appears to allosterically inhibit the hyaluronidase activity. Main sugar composition and sulfate content of SP was estimated to be Gal, Glc, Xyl, Man, uronic acids, and sulfate in the weight percent of 27.7: 28.9: 14.6: 22.5: 3.4: 21.7. We modified the SP by desulfation and partial hydrolysis with trifluoroacetic acid (TFA) to investigate the effect of sulfate content and molecular weight on inhibition. Hyaluronidase inhibitory activity of desulfated SP, 0.1 M TFA-hydrolyzed SP and 0.5 M TFA-hydrolyzed SP were significantly lower than that of native SP, revealing that sulfate content or molecular weight is important for hyaluronidase inhibition.