Regulation of sialic acids by human neuraminidase (hNEU) enzymes is important to many biological processes. Defining hNEU substrate tolerance can help to elucidate the roles of these enzymes in regulating sialosides in human health and disease. Polysialic acid (polySia) is a polyanion of α(2→8) linked sialic acids with roles in nervous, reproductive, and immune systems and is dysregulated in some malignancies and mental disorders. The unique chemical properties of this polymer, which include an enhanced susceptibility to acid-catalyzed hydrolysis, have hampered its study. Herein we describe the first systematic study of hNEU isoenzyme activity towards polysialic acid in vitro. The experimental design allowed us to study the impact of several factors that may influence polysialic acid degradation including pH, polymer size, and the relative ionic strength of the surrounding media. We report that short chains of polysialic acid (degree of polymerization, DP 3-8) were substrates of NEU3 and NEU4 at acidic pH, but not at neutral pH. No hNEU-catalyzed hydrolysis of longer polymers (DP 10-20) was detected. These findings suggest a neuraminidase-independent mechanism for polysialic acid turnover such as internalization and degradation in endosomes and lysosomes.