Expression of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα) in resting lymphocytes was recently established, although the physiologic role (s) played by this nuclear hormone receptor in these cell types remains unresolved. In this study, we used CD4+ T cells isolated from PPARα−/− and wild-type mice, as well as cell lines that constitutively express PPARα, in experiments designed to evaluate the role of this hormone receptor in the regulation of T cell function. We report that activated CD4+ T cells lacking PPARα produce increased levels of IFN-γ, but significantly lower levels of IL-2 when compared with activated wild-type CD4+ T cells. Furthermore, we demonstrate that PPARα regulates the expression of these cytokines by CD4+ T cells in part, through its ability to negatively regulate the transcription of T-bet. The induction of T-bet expression in CD4+ T cells was determined to be positively influenced by p38 mitogen-activated protein (MAP) kinase activation, and the presence of unliganded PPARα effectively suppressed the phosphorylation of p38 MAP kinase. The activation of PPARα with highly specific ligands relaxed its capacity to suppress p38 MAP kinase phosphorylation and promoted T-bet expression. These results demonstrate a novel DNA-binding independent and agonist-controlled regulatory influence by the nuclear hormone receptor PPARα.