In the present study we describe the isolation and characterization of putative equine granzyme B for which we propose the designation 'eqGrzmB'. Sequence analysis revealed characteristic features of a GrzmB protease such as the presence of a signal (leader-) peptide and an activation di-peptide. The isolated eqGrzmB is functionally active when expressed in human or in insect cells. Furthermore, exchange of any of three putative active site amino acids, which are highly conserved along granzyme B enzymes, led to a complete loss of enzymatic activity in the newly identified eqGrzmB. Phylogenetic analysis places eqGrzmB in the chymase-locus within the large family of granzymes in close proximity to putative equine mast cell protease and to granzyme B from mouse, rat, and human. eqGrzmB proteolytic activity has been kinetically characterized and can be specifically inhibited by granzyme B inhibitors. Taken together, we conclude that we have isolated a new member of the granzyme B family, the first granzyme identified in Equidae. The description of equine granzyme B might facilitate the development of immunological assays for the activity of equine lymphocytes.