Haemophilus parasuis， the causative agent of Glasser's disease， is a pathogen that colonizes the upper respiratory tract (URT) of pigs， invades the bloodstream and causes polyserositis. Because antimicrobials are highly effective against H. parasuis， we hypothesized that they could have a detrimental effect on the establishment of an immune response if given at the time of URT colonization. In this study， we characterized clinical outcomes and antibody and IFN-γ responses to H. parasuis in pigs treated with enrofloxacin before or after low dose inoculation with a pathogenic H. parasuis strain. Pigs that were only inoculated with the agent (EXP group) and pigs that were treated with enrofloxacin and then inoculated (ABT/EXP group) developed signs of disease starting at 4days post inoculation (DPI)， presented a significant increase in serum IgG and were protected against a subsequent homologous challenge. In contrast， pigs treated with antibiotic after inoculation (EXP/ABT group) neither showed signs of disease nor seroconverted (IgG) after low dose inoculation. EXP/ABT pigs as well as na?ve control pigs [enrofloxacin only (ABT) and challenge only (CHA)] were susceptible to challenge. Variable levels of antibodies in bronchioalveolar fluid and IFN-γ in peripheral blood mononuclear cells were observed after H. parasuis inoculation， but were not associated with protection. In summary， only pigs treated before low dose H. parasuis inoculation seroconverted and were protected against subsequent challenge. Results from this study can help determine timing of antimicrobial use and contribute to our current understanding of judicious antibiotic use.