Granzyme B (GrB) is a serine protease that is released by cytotoxic lymphocytes to kill virus-infected and tumor cells. Recent advances in the understanding of GrB have stressed the importance of reassessing the mechanisms by which GrB accomplishes its death functions. These include the uptake and trafficking of GrB within target cells, pathways used to trigger cell death, and the mechanism(s) controlling its killing activity. In addition, the role that GrB plays in human pathologies is still to be defined. The purpose of this review is to discuss recent insights into the biology of GrB and to evaluate its functional significance in health and disease.