In poultry, Escherichia coli is a common cause of high-cost infections. Consequently, au-togenous vaccines are often used despite limited and conflicting evidence on their effectiveness have been presented. The present study aimed to investigate the efficacy of a commonly used autogenous vaccine, previously deemed ineffective, in an aerosol model of colibacillosis. Methods: Broiler breeders (n = 47) were randomly allocated to one of four groups (vaccinated and unvaccinated birds receiving an autogenous vaccine or sterile saline intramuscularly) and challenged with either aerosolised E. coli or vehicle at 29 weeks of age. Two days following inoculation, the birds were euthanised, thoroughly necropsied, and samples for bacteriology and histopathology were collected. Results: Vaccinated birds had a significantly lower bacteriology score compared to the unvaccinated group challenged with E. coli (p < 0.01) and a lower overall air sac lesion score (p < 0.05). Overall lung and spleen lesion scores only differed significantly between the unvaccinated E. coli challenged group compared to the vehicle inoculated groups. The overall gross pathology score was 2.8 and 1.95 in the unvaccinated and vaccinated E. coli challenge groups, respectively, whereas the vaccinated vehicle group had a score of 0.9 and the unvaccinated vehicle group a score of 1. Conclusions: A protective effect of an autogenous vaccine was found utilising an aerogenous model of colibacillosis through multiple methods of evaluation. The findings encourage the continued use of autogenous vaccines and underlines the necessity of discriminative experimental models with high predictive validity when evaluating vaccine interventions.