Peroxisome proliferator-activated receptor (PPAR) agonists are promising new agents for treatment of the metabolic syndrome. Although they possess antiatherosclerotic properties in vivo and promote endothelial cell survival, their mechanism of action is incompletely understood. 14-3-3 is a critical component of the endothelial cell antiapoptotic machinery, which is essential to maintain homeostasis of the vascular wall. To test the hypothesis that PPAR targets 14-3-3 in endothelial cells, we studied the response of the gene that encodes 14-3-3 in humans, YWHAE, to PPAR ligands (L-165,041 and GW501516). We found that PPAR activates YWHAE promoter in a concentration and time-dependent manner. Consistent with these findings, L-165,041 increased 14-3-3 mRNA and protein level, whereas PPAR small interfering RNA suppressed both basal and L-165,041-dependent YWHAE transcription and 14-3-3 protein expression. Surprisingly, PPAR response elements in YWHAE promoter were not required for upregulation by PPAR, whereas a CCAAT/enhancer binding protein (C/EBP) site located at -160/-151 bp regulated both basal and PPAR-dependent promoter activity. Intriguingly, activation or knock down of endogenous PPAR regulated C/EBP protein expression. Chromatin immunoprecipitation assays demonstrated that L-165,041 determines the localization of C/EBP to the region spanning this C/EBP response element, whereas sequential chromatin immunoprecipitation analysis revealed that C/EBP and PPAR form a transcriptional activating complex on this C/EBP site. Our work uncovers a novel role for C/EBP as a mediator of PPAR-dependent 14-3-3 gene regulation in human endothelial cells and provides insight into the mechanism by which PPAR agonists may be beneficial in atherosclerosis.