Vibrio cholerae neuraminidase (VCNA) plays a significant role in the pathogenesis of cholera by removing sialic acid from higher order gangliosides to unmask GM1, the receptor for cholera toxin. We previously showed that the structure of VCNA is composed of a central ?-propeller catalytic domain flanked by two lectin-like domains, however the nature of the carbohydrates recognised by these lectin domains has remained unknown. We present here structures of the enzyme in complex with two substrates, ?2,3-sialyllactose and ?-2,6-sialyllactose. Both substrate complexes reveal the ?-anomer of N-acetylneuraminic acid (Neu5Ac, NANA) bound to the Nterminal lectin domain, thereby revealing the role of this domain. The large number of interactions suggest a relatively high binding affinity for sialic acid, which was confirmed by calorimetry, which gave a Kd~30?M. Saturation transfer difference (STD) NMR using a non-hydrolysable substrate, Neu5,9Ac2-2-S-(?-2,6)-GlcNAc?1Me, was also used to map the ligand interactions at the VCNA lectin binding site. It is well known that VCNA can hydrolyse both ?-2,3- and ?-2,6-linked sialic acid substrates. In this study using ?-2,3-sialyllactose co-crystallised with VCNA it was revealed that the inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, DANA) was bound at the catalytic site. This observation supports the notion that VCNA can produce its own inhibitor and has been further confirmed by 1H NMR analysis. The discovery of the sialic acid-binding site in the N-lectin-like domain suggests that this might help target VCNA to sialic acid-rich environments, thereby enhancing the catalytic efficiency of the enzyme.