Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) expression and activation is involved in the cell proliferation. However, little is known about the role of PPARbeta/delta in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPARbeta/delta mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPARbeta/delta protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPARbeta/delta binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPARbeta/delta caused selectively inhibition of PPARbeta/delta protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPARbeta/delta, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPARbeta/delta up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPARbeta/delta promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPARbeta/delta expression in a c-Jun-dependent manner and PPARbeta/delta plays a vital role in EGF-stimulated proliferation of HaCaT cells.