Neisseria meningitidis resides normally harmlessly in the human nasopharynx, but can cause fatal sepsis and meningitis worldwide. Moonlighting proteins are a group of proteins which can perform several autonomous, unrelated functions when localised at different sites. They have been increasingly reported on the surface of both prokaryotic and eukaryotic organisms and shown to interact with a variety of host ligands and perform virulence-related functions. Three enzymes of Embden-Meyerhof-Parnas (EMP) glycolytic pathway (an inactive pathway in N. meningitidis) namely enolase, glyceraldehyde 3- phosphate dehydrogenase (GAPDH), and fructose 1, 6 bisphosphate aldolase (FBA) have been shown to be localised to the meningococcal cell surface and to have non-glycolytic (moonlighting) functions related to interactions with host proteins or adhesion to host cells. This study further explores the moonlighting functions of FBA in the pathogenesis of meningococcal disease. Recombinant wild-type FBA and FBA with mutations in the active cation-binding site (D83A and H81A/H84A) were cloned, overexpressed and purified under non-denaturing condition. A coupled enzyme assay confirmed the aldolase activity of wild-type rFBA. In contrast, rFBA with mutation(s) in the active (cation-binding) site (D83A and H81A/H84A) had no detectable enzymatic activity. Employing flow cytometry, FBA could be detected on the surface of wild type N. meningitidis MC58 cells but not on MC58ΔcbbA (FBA-deficient mutant). Complementation of MC58ΔcbbA with an ectopic copy of cbbA (either wild type or D83A and H81A/H84A variants) restored the ability to express FBA on the surface suggesting the lack of involvement of the active site in the transportation of FBA to the cell surface. Moreover, N. meningitides MC58ΔcbbA showed impaired adherence to human brain endothelial (HBME) cells compared with its wild type parent. Complementation of MC58ΔcbbA with an ectopic copy of cbbA (either wild type, or D83A and H81A/H84A variants) restored the ability to adhere to HBME cells. Furthermore, rFBA was shown to bind human plasminogen. No significant difference was observed between the plasminogen binding by wild type rFBA and rFBA lacking aldolase activity. Plasminogen binding was inhibited by the lysine analogue, Ɛ-aminocaproic acid, indicating the involvement of lysine residue(s) in this interaction. A truncated rFBA comprising the C-terminal 134 amino acids and containing several lysine-rich motifs was shown to bind plasminogen as well as the wild type rFBA. Substitution of the terminal lysine residue of rFBA with alanine dramatically reduced the binding of plasminogen. Moreover, both pathogenic and non-pathogenic neisserial species were shown to bind human plasminogen. Taken together, our data suggest that the moonlighting functions of meningococcal FBA on the bacterial surface include the binding of human plasminogen and the facilitation of optimal adhesion to host cells, and that both of these phenotypes are independent of its aldolase activity.