Recognition of terminal sialic acids is central to many cellular processes, and structural modification of sialic acid can disrupt these interactions. A prominent, naturally occurring, modification of sialic acid is 9- O-acetylation (9- O-Ac). Study of this modification through generation and analysis of 9- O-Ac sialosides is challenging because of the lability of the acetate group. Fundamental questions regarding the role of 9- O-Ac sialic acids remain unanswered, including what effect it may have on recognition and hydrolysis by the human neuraminidase enzymes (hNEU). To investigate the substrate activity of 9- O-acetylated sialic acids (Neu5,9Ac2), we synthesized an acetylated fluorogenic hNEU substrate 2'-(4-methylumbelliferyl)-9- O-acetyl-α-d- N-acetylneuraminic acid. Additionally, we generated a panel of octyl sialyllactosides containing modified sialic acids including variation in linkage, 9- O-acetylation, and C-5 group (Neu5Gc). Relative rates of substrate cleavage by hNEU were determined using fluorescence spectroscopy and electrospray ionization mass spectrometry. We report that 9- O-acetylation had a significant, and differential, impact on sialic acid hydrolysis by hNEU with general substrate tolerance following the trend of Neu5Ac > Neu5Gc ≫ Neu5,9Ac2 for NEU2, NEU3, and NEU4. Both NEU2 and NEU3 had remarkably reduced activity for Neu5,9Ac2 containing substrates. Other isoenzymes appeared to be more tolerant, with NEU4 even showing increased activity on Neu5,9Ac2 substrates with an aryl aglycone. The impact of these minor structural changes to sialic acid on hNEU activity was unexpected, and these results provide evidence of the substantial influence of 9- O-Ac modifications on hNEU enzyme substrate specificity. Furthermore, these findings may implicate hNEU in processes governed by 9- O-acetyltransferases and -esterases.