Epidermal growth factor (EGF) plays an important role in epithelial cell proliferation and apoptosis. Our recent studies found that EGF-attenuated tumor necrosis factor-alpha induced HaCaT keratinocyte apoptosis, and this effect was accompanied by up-regulation of the expression of peroxisome proliferator-activated receptor beta (PPARbeta). However, little is known about whether PPARbeta is functionally involved in the inhibition of keratinocyte apoptosis by EGF. Here, we showed that EGF up-regulated the DNA-binding and transcriptional regulation activities of PPARbeta. Antisense phosphorothioate oligonucleotides against PPARbeta markedly inhibited de novo synthesis of PPARbeta and attenuated the protective effect of EGF on tumor necrosis factor-alpha-induced apoptosis. L165041, a specific PPARbeta ligand, significantly enhanced the transcriptional regulation activity of PPARbeta and increased the protective effect of EGF. These results suggest a molecular mechanism by which EGF protects HaCaT keratinocytes against apoptosis in a PPARbeta-dependent manner.