The aim of the present study was to evaluate the role of endogenous and exogenous peroxisome proliferator-activated receptor α (PPAR-α), a nuclear receptor, on the regulation of inflammation in macrophages. To address this question, we have stimulated peritoneal macrophages from PPAR-α wild-type mice and PPAR-α knockout mice (PPAR-α) with 10 μg/mL LPS and 100 U/mL IFN-γ. We report here that the absence of a functional PPAR-α gene in PPAR-α knockout mice resulted in a significant augmentation of various inflammatory parameters in peritoneal macrophages. In particular, we have clearly demonstrated that PPAR-α gene deletion increases (1) the mitogen-activated protein kinase phosphorylation (extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38), (2) nuclear factor-κB activation, (3) IκB-α degradation, (4) iNOS expression and NO formation, and (5) cyclooxygenase 2 expression and prostaglandin E2 formation caused by LPS/IFN-γ stimulation. On the contrary, the incubation of peritoneal macrophages from PPAR-α wild type with clofibrate (2 mM) at 2 h before the LPS and IFN-γ stimulation significantly reduced the expression and the release of the proinflammatory mediators. To elucidate whether the protective effects of clofibrate is related to activation of the PPAR-α receptor, we also investigated the effect of clofibrate treatment on PPAR-α-deficient mice. The absence of the PPAR-α receptor significantly abolished the protective effect of the PPAR-α agonist against LPS/IFN-γ-induced macrophage inflammation. In conclusion, our study demonstrates that the endogenous and exogenous PPAR-α ligands reduce the degree of macrophage inflammation caused by LPS/IFN-γ stimulation.