Background: Natural immunity to Neisseria meningitidis may result from nasopharyngeal carriage of closely related commensals, such as Neisseria lactamica. Methods: We enrolled 61 students with no current carriage of Neisseria species and inoculated them intranasally with 10,000 colony-forming units of Neisseria lactamica or sham control. Colonization was monitored in oropharyngeal samples over 6 months. We measured specific mucosal and systemic antibody responses to N. lactamica and serum bactericidal antibody (SBA) and opsonophagocytic antibodies to a panel of N. meningitidis serogroup B strains. We also inoculated an additional cohort following vaccination with N. lactamica outermembrane vesicles (OMV) produced from the same strain. Results: Twenty-six (63.4%) of 41 inoculated individuals became colonized with N. lactamica; 85% remained colonized at 12 weeks. Noncarriers were resistant to rechallenge, and carriers who terminated carriage were relatively resistant to rechallenge. No carriers acquired N. meningitidis carriage over 24 weeks, compared with 3 control subjects (15%). Carriers developed serum IgG and salivary IgA antibodies to the inoculated N. lactamica strain by 4 weeks; noncarriers and control subjects did not. Cross-reactive serum bactericidal antibody responses to N.meningitidis were negligible in carriers, but they developed broad opsonophagocytic antimeningococcal antibodies. OMV vaccinees developed systemic and mucosal anti-N. lactamica antibodies and were relatively resistant to N. lactamica carriage but not to natural acquisition of N. meningitidis. Conclusions: Carriers of N. lactamica develop mucosal and systemic humoral immunity to N. lactamica together with cross-reacting systemic opsonophagocytic but not bactericidal antibodies to N. meningitidis. Possession of humoral immunity to N. lactamica inhibits acquisition of N. lactamica but not of N. meningitidis. Some individuals are intrinsically resistant to N. lactamica carriage, independent of humoral immunity.