Granzyme B (GrB) is an immune protease implicated in the pathogenesis of several human diseases. In the current model of GrB activity, perforin determines whether the downstream actions of GrB occur intracellularly or extracellularly, producing apoptotic cytotoxicity or nonapoptotic effects, respectively. In the current study, we demonstrate the existence of a broad range of GrB-dependent signaling activities that 1) do not require perforin, 2) occur intracellularly, and 3) for which cell death is not the dominant outcome. In the absence of perforin, we show that GrB enzymatic activity still induces substoichiometric activation of caspases, which through nonlethal DNA damage response signals then leads to activity-associated phosphorylation of IFN regulatory factor-3. These findings illustrate an unexpected potential interface between GrB and innate immunity separate from the traditional role of GrB in perforin-dependent GrB-mediated apoptosis that could have mechanistic implications for human disease.