The granule-exocytosis pathway is the main mechanism via which cytotoxic lymphocytes exert their anti-viral and anti-tumor functions. Upon recognition of a target cell, activated immune cells deliver a set of homologous serine proteases known as granzymes inside the target cell, where they can exert their intracellular functions. One of these granzymes, granzyme M (GrM), induces cell death in tumor cells and protects against cytomegalovirus (CMV) infection. However, the molecular mechanisms of GrM actions remain poorly understood and are further elucidated in the present thesis. First, we comprehensively characterize GrM-induced cell death in tumor cells. We show that GrM triggers cell cycle arrest and caspase-dependent apoptosis by cleaving the nuclear enzyme DNA topoisomerase II alpha. Second, we identify SERPINB4 as an intracellular inhibitor of GrM that can inhibit its pro-apoptotic function in tumor cells. Third, we demonstrate that GrM exerts a noncytotoxic anti-viral function by targeting host cell protein heterogeneous nuclear ribonucleoprotein K to block CMV replication. Fourth, we identified several species-specific differences between human GrM and its mouse ortholog, which has major implications for the use of GrM-knockout mouse models. Lastly, we have developed a novel bioluminescent sensor to detect intracellular GrM activity, which may be used to investigate the physiological role of GrM in vitro and in vivo. Overall, our findings result in a better understanding of how immune cells fulfil their (non)cytotoxic anti-tumor and anti-viral functions, which may eventually lead to novel therapeutic approaches.