Granzyme B expression is essential for eliciting NK cell cytotoxicity and T cell function. However, its transcriptional regulatory mechanism is not well understood. In this report, we demonstrate in human NK cells and T cells that the NF-kappaB-signaling pathway is involved in such control. Furthermore, a novel downstream human granzyme B gene sequence (GGAGATTCCC) was identified for NF-kappaB binding. EMSA, luciferase, and chromatin immunoprecipitation assays in vitro and in vivo indicated that this NF-kappaB binding site is functional in an NK cell line and its primary counterpart. Our data also demonstrate that this binding site is functional in Jurkat T cells. Taken together, we identified a novel NF-kappaB binding site, which plays a pivotal role in controlling human granzyme B gene transcription.