Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors (NRs) that regulate genes involved in lipid and glucose metabolism. PPAR activity is regulated by interactions with cofactors and of interest are cofactors with ubiquitin ligase activity. The E6-associated protein (E6-AP) is an E3 ubiquitin ligase that affects the activity of other NRs, although its effects on PPARs have not been examined. E6-AP inhibited the ligand-independent transcriptional activity of PPARalpha and PPARbeta, with marginal effects on PPARgamma, and decreased basal mRNA levels of PPARalpha target genes. Inhibition of PPARalpha activity required the ubiquitin ligase function of E6-AP, but occurred in a proteasome-independent manner. PPARalpha interacted with E6-AP, and in mice treated with PPARalpha agonist clofibrate, mRNA and protein levels of E6-AP were increased in wildtype, but not in PPARalpha null mice, indicating a PPARalpha-dependent regulation. These studies suggest coordinate regulation of E6-AP and PPARalpha, and contribute to our understanding of the role of PPARs in cellular metabolism.