A primary cause of acute otitis media (AOM) in humans is non-typeable Haemophilus influenzae (NTHi). AOM is the most common bacterial infection in children and reason for antibiotic prescription to this age group. The mouse has been preferentially used as a model organism for human disease for many years and we have developed a robust middle ear infection model in the Junbo mouse, a mutant mouse line that develops symptoms of otitis media spontaneously in specific pathogen free conditions. The heterozygote Junbo mouse bears a mutation in the gene encoding the Evi1 transcription factor that is a negative regulator of NFkB. Experimental challenge through a single intranasal dose with NTHi produces at day 7 post-inoculation significant rates of middle ear infection (30 to 95%) and bacterial titers (up to 105 c.f.u./µl) in middle ear bulla fluids that can remain infected up to at least 56 days post-inoculation. Dual infection using a pair of NTHi strains indicated that at day 7 post-inoculation mouse middle ears predominantly contain mono-culture of one or the other strain whereas most ears have mixed NTHi culture at day one. The translational potential of the Junbo model for immunization and antibiotics treatment strategies has been demonstrated. Treatment of infected mice with a 3 day course of Azithromycin cleared NTHi infection and immunization of mice with killed NTHi bacteria resulted in protection against challenge with the homologous, but not heterologous, NTHi strain.