Membrane-bound sialidase Neu3 is involved in the catabolism of glycoconjugates, and plays crucial roles in numerous biological processes. Since the mechanism of its association with membranes is still not completely understood, the aim of this work was to provide further information regarding this aspect. Human Neu3 was found to be associated with the plasma membrane and endomembranes, and it was not released from the lipid bilayer under conditions that typically release peripheral membrane proteins. By different experimental approaches, we demonstrated that its C-terminus is exposed to the cytosol while another portion of the protein is exposed to the extracellular space, suggesting that Neu3 possesses the features of a transmembrane protein. However, in silico analysis and homology modeling predicted that the sialidase does not contain any α-helical transmembrane segment and shares the same β-propeller fold typical of viral and bacterial sialidases. Additionally, we found that Neu3 is S-acylated. Since this post-translational modification is restricted to the cytosolic side of membranes, this finding strongly supports the idea that Neu3 may contain a cytosolic-exposed domain. Although it remains to be determined exactly how this sialidase crosses the lipid bilayer, this study provides new insights about membrane association and topology of Neu3.