CD4+ T cell activation and differentiation are important events that set the stage for proper immune responses. Many factors are involved in the activation and differentiation of T cells, and these events are tightly controlled to prevent unwanted and/or exacerbated immune responses that may harm the host. It has been well-documented that granzyme B, a potent serine protease involved in cell-mediated cytotoxicity, is readily expressed by certain CD4+ T cells, such as regulatory T cells and CD4+CD8αα+ intestinal intraepithelial lymphocytes, both of which display cytotoxicity associated with granzyme B. However, because not all CD4+ T cells expressing granzyme B are cytotoxic, additional roles for this protease in CD4+ T cell biology remain unknown. Here, using a combination of in vivo and in vitro approaches, we report that granzyme B-deficient CD4+ T cells display increased IL-17 production. In the adoptive transfer model of intestinal inflammation, granzyme B-deficient CD4+ T cells triggered a more rapid disease onset than their WT counterparts, and presented a differential transcription profile. Similar results were also observed in granzyme B-deficient mice infected with Citrobacter rodentium. Our results suggest that granzyme B modulates CD4+ T cell differentiation, providing a new perspective into the biology of this enzyme.