Native outer membrane vesicles (NOMV) (not detergent treated), which are prepared from recombinant strains with attenuated endotoxin activity and overexpressed factor H binding protein (fHbp), elicited broad serum bactericidal antibody responses in mice. The amount of overexpressed fHbp required for optimal immunogenicity is not known. In this study we prepared NOMV vaccines from LpxL1 knockout (ΔLpxL1) mutants with penta-acylated lipooligosaccharide and attenuated endotoxin activity. The recombinant strains had wild-type (1×) fHbp expression or were engineered for 3-fold- or 10-fold-increased fHbp expression (3× or 10× fHbp). Control vaccines included NOMV from ΔLpxL1/ΔfHbp mutants or recombinant fHbp. In mice, only the 10× fHbp NOMV vaccine elicited significantly higher serum IgG anti-fHbp antibody titers than the corresponding 1× fHbp NOMV or recombinant fHbp vaccine. The 10× fHbp NOMV vaccine also elicited higher bactericidal responses (P < 0.05) against five group B strains with heterologous PorA than the recombinant fHbp or 1× fHbp NOMV vaccine. The 3× fHbp NOMV vaccine gave higher bactericidal titers against only one strain. Serum bactericidal titers in mice immunized with the control ΔfHbp NOMV vaccines were <1:10, and bactericidal titers in mice immunized with the 10× fHbp NOMV vaccine were <1:10 after adsorption of anti-fHbp antibodies. Mixing antiserum to NOMV vaccines from fHbp knockout mutants with antiserum to recombinant fHbp did not increase anti-fHbp bactericidal titers. Thus, a critical threshold of increased fHbp expression is required for NOMV vaccines to elicit broad serum bactericidal responses, and the antibodies conferring protection are directed primarily at fHbp.