The aim of this study was to evaluate the effect of the excipients used to facilitate the encapsulation of high hydrophobic antigenic complex extracted from Brucella ovis (HS) on the physico-chemical properties of the resulting microparticles. Poly(epsilon-caprolactone) (PEC) microparticles containing HS were prepared by the solvent extraction/evaporation method using total recirculation one-machine system (TROMS). Different excipients, beta-cyclodextrin (beta-CD), Pluronic F68, Tween 20 or Tween 80, were used in order to facilitate the encapsulation and conserve the bioactivity of the encapsulated antigenic complex. HS was efficiently loaded in all the different PEC-microparticle formulations, although the combined use of beta-cyclodextrin and Pluronic F68 permitted an increase in the amount of antigenic extract in the core of the resulting microparticles without loss of its antigenic properties. Finally, the protective ability of this F68-CD-MP formulation was evaluated against an experimental challenge with the virulent Brucella melitensis H38 strain in BALB/c mice. This innocuous subcellular vaccine induced a similar protection to that of the live attenuated Rev 1 vaccine; these are promising results that would merit further investigation in target animals.