Substitution of Neisseria gonorrhoeae lacto-N-neotetraose (LNT) lipooligosaccharide (LOS) with Neu5Ac confers on the bacterium the ability to evade complement-mediated killing by enhancing binding of the alternative pathway inhibitor, factor H (FH), and also reducing binding of specific IgG. We utilized the CMP salts of six sialic acid analogues, Neu5Gc, Neu5Ac9Ac, Neu5Ac9Az, Neu5Gc8Me, Leg5Ac7Ac and Pse5Ac7Ac, to characterize the substrate specificity for gonococcal LOS sialyltransferase (Lst) and define the structural requirements of sialic acid-mediated complement resistance. All analogues, except Pse5Ac7Ac, served as substrates for gonococcal Lst and were incorporated on to LNT LOS. Only Neu5Gc incorporation yielded FH binding and high-level serum resistance comparable with Neu5Ac-substituted LOS. Neu5Ac9Ac and Neu5Gc8Me permitted resistance only to low, but not high complement concentrations, while Neu5Ac9Az and Leg5Ac7Ac did not confer any resistance to complement. Neu5Ac9Ac addition resulted in low-level FH binding (~20% of the levels seen with Neu5Ac and Neu5Gc), but addition of none of the remaining analogues showed detectable FH binding by FACS. The extent of classical pathway inhibition (measured by IgG binding and C4 deposition) was proportional to serum resistance. Adding CMP-Neu5A9Az or CMP-Leg5Ac7Ac to bacteria within 15 min of adding CMP-Neu5Ac did not block FH binding facilitated by Neu5Ac, but blocked classical pathway suppression and serum resistance mediated by Neu5Ac on strain F62 and a high-level ceftriaxone-resistant isolate called H041, even when Neu5Ac was present at a 10- 100-fold molar excess. Administration of CMP-Leg5Ac7Ac intravaginally significantly reduced the duration of infection (median time to clearance 6 and 10 days in treated and untreated mice, respectively; P<0.0001) and bacterial burdens (P=0.0005) in the murine vaginal colonization model of gonorrhea. Collectively, these data shed light on substrate specificity of gonococcal Lst and reveal critical roles for carbon 7, 8 and 9 substitutions on sialic acid for complement inhibition on gonococci. The use of sialic acid analogues to counter gonococcal virulence provides a novel therpeutic strategy against the global threat of multi-drug resistant gonorrhea.