Mannheimia haemolytica (M. haemolytica) causes significant losses in livestock, but cross-protection between serotypes is limited. Current commercial vaccines primarily target serotypes 1 (S1) and 2 (S2) despite the increasing incidence of serotype 6 (S6) infections. While leukotoxin (LKT) is a common vaccine target, serotype-1 specific antigen (SSA-1) is often overlooked. Furthermore, neutralizing antibody (nAb) titers, crucial for evaluating vaccine efficacy, are not routinely measured. This study aimed to develop a trivalent vaccine targeting S1, S2, and S6 using recombinant LKT (rLKT) and rSSA-1, and to evaluate total IgG and nAbs responses following vaccination in the murine model. Three M. haemolytica strains (S1, S2, and S6) with diverse phenotypic characteristics were selected. A host specificity protein J (250 kDa) was identified in the S6 strain grown in Todd-Hewitt broth. This protein caused widespread bleeding in experimental mouse groups, raising considerations for its inclusion in future vaccine formulations. A trivalent vaccine was prepared by different serotypes (S1, S2, and S6), rLKT, rSSA-1, and Montanide™ ISA 206 VG adjuvant. Mice were vaccinated twice at 21-day intervals. Total IgG and nAb titers were measured using in-house ELISAs and Vero cell neutralization assays, respectively. Total IgG revealed the highest antibody responses against S2 pellet and S6 supernatant antigens. The result of nAb titers in the vaccinated mice; was 1/80 (log101.9) against three pellets (S1, S2, and S6), and supernatant protein (S6) in contrast to 1/40 (log101.6) against other supernatant proteins (S1, S2). The vaccine demonstrated an odds ratio of 0.97. Although total IgG titers against S1 were lower compared to other serotypes, nAb increases were similar across all serotypes, highlighting the importance of measuring nAb titers in addition to total IgG for a comprehensive vaccine evaluation. Challenge studies further corroborated the stimulation of nAbs. The trivalent vaccine effectively stimulated both total IgG and nAb responses against all three serotypes in mice, suggesting its potential for broad protection against M. haemolytica.