Eosinophils are known to play a crucial role in equine insect bite hypersensitivity (IBH), an allergic and pruritic dermatitis. Given the relevant role of eosinophils in disease pathology, a virus-like particle (VLP)-based vaccination targeting equine Interleukin (eIL)-5 has been shown to significantly reduce eosinophil levels in the blood and hence reduced IBH symptoms in horses. The aim of the present study was to assess the presence of different eosinophil subtypes in horses, describe characteristics of these subtypes and evaluate the effect of eIL-5-CuMVTT vaccination on these subtypes. The study involved healthy horses, untreated IBH horses, eIL-5-CuMVTT vaccinated and placebo-treated IBH horses, assessed in parallel during the same IBH season. For the first time, this study showed presence of two distinct eosinophil subtypes in horses. An inflammatory subtype was associated with allergy, which had a significantly larger cell size, increased intracellular granula and higher surface expression of the integrin CD49f ^hi. Regulatory steady-state eosinophils were identified in healthy blood sample and with smaller relative cell size, had less intracellular granula and significantly lower levels of surface integrin CD49f ^dim. The eIL-5-CuMVTT vaccination significantly reduced eosinophil levels in the blood throughout the whole IBH season, indicating IL-5-dependence of inflammatory eosinophils. However, it did not alter the phenotype of the remaining cells. The mechanism of action of vaccination likely acts by limiting availability of eosinophils in the blood for extravasation into the skin.