BACKGROUND: Peroxisome proliferator-activated receptor α (PPARα) plays a crucial role in liver physiological and pathological processes. Yes-associated protein (YAP) is a key effector in regulating cell growth and organ size. Ubiquitination is known to modulate YAP protein expression, stability, and nuclear localization. Our previous study demonstrated that PPARα activation promotes hepatomegaly and liver regeneration via YAP activation. However, the underlying molecular mechanisms by which PPARα regulates YAP are unclear. In this study, PPARα was activated by the classical agonist WY-14643, and its effects on YAP ubiquitination were examined using plasmid transfection and immunoprecipitation. The ubiquitination of YAP was further investigated through mutant YAP plasmids, gene knockdown, and immunofluorescence staining. YAP mRNA and protein expression were measured via qRT-PCR and western blotting. RESULTS: The results demonstrated that PPARα activation upregulated YAP protein levels and enhanced its activity, while reducing overall YAP ubiquitination. Specifically, PPARα activation inhibited K48-linked ubiquitination while promoting K63-linked ubiquitination of YAP. Mutations at the K252, K321, and K497 residues of YAP markedly reduced the capacity of PPARα activation to facilitate YAP nuclear translocation. Furthermore, knockdown of the E3 ligase TRAF6 abolished the PPARα-induced K63-linked ubiquitination of YAP and the upregulation of its downstream target genes. CONCLUSIONS: These findings highlight the pivotal role of ubiquitination in regulating YAP through PPARα activation, providing novel insights for future studies on the post-translational regulation of YAP by PPARα activation.