Beauveria bassiana is widely studied as an alternative to chemical acaricides in controlling the cattle tick Rhipicephalus microplus. Although its biocontrol efficiency has been proved in laboratory and field scales, there is a need to a better understanding of host interaction process at molecular level related to biocontrol activity. In this work, applying a proteomic technique multidimensional protein identification technology (MudPIT), the differential secretome of B. bassiana induced by the host R. microplus cuticle was evaluated. The use of the host cuticle in a culture medium, mimicking an infection condition, is an established experimental model that triggers the secretion of inducible enzymes. From a total of 236 proteins, 50 proteins were identified exclusively in infection condition, assigned to different aspects of infection like host adhesion, cuticle penetration and fungal defense, and stress. Other 32 proteins were considered up- or down-regulated. In order to get a meaningful global view of the secretome, several bioinformatic analyses were performed. Regarding molecular function classification, the highest number of proteins in the differential secretome was assigned in to hydrolase activity, enzyme class of all cuticle-degrading enzymes like lipases and proteases. These activities were also further validated through enzymatic assays. The results presented here reveal dozens of specific proteins and different processes potentially implicated in cattle tick infection improving the understanding of molecular basis of biocontrol of B. bassiana against R. microplus.