Knowledge on the characteristics of different infection models of avian colibacillosis is mandatory for performing reliable experimental studies. This study compares the characteristics of two infection models of colibacillosis (by intratracheal and subcutaneous routes) in broilers. Broilers (125), 35 days old, were randomly allocated to four experimental (n = 20) and three control (n = 15) groups: 1) negative control; 2) intratracheal infection control; 3) subcutaneous infection control; 4) intratracheal infection (IT); 5) intratracheal infection with florfenicol administration (ITF); 6) subcutaneous infection (SC); and 7) subcutaneous infection with florfenicol administration (SCF). Clinical signs and mortalities were recorded, and gross examination of carcasses was performed. On days 3 and 5 postchallenge (PC), 5 birds/group were euthanatized. Despite similar onset and type of clinical signs and mortality of birds in the IT and SC groups, especially on early days PC, birds in the SC group had lower body weight than IT birds. Lung injuries were more prominent in the IT group than the SC group. Birds of the SC group showed cellulitis and severely injured kidneys in contrast to the IT group. On day 3 PC, the number of positive heart blood samples for bacterial growth in the ITF group was half that of the IT group, whereas SC and SCF birds had similar positive numbers. Infection by the subcutaneous route results in more severe disease. The intratracheal route produces results more similar to colibacillosis under field conditions with regard to clinical signs, pathogenesis, and response to therapy, albeit it is more difficult to perform than a subcutaneous challenge.