The natural cofactor of phenylalanine hydroxylase (PAH), tetrahydrobiopterin (BH₄), regulates the enzyme activity as well as being essential in catalysis. BH₄-responsive PAH deficiency is a variant of hyperphenylalaninemia or phenylketonuria (PKU) caused by mutations in the human PAH gene that respond to oral BH₄ loading by stimulating enzyme activity and therefore lowering serum phenylalanine. Here, we showed in a coupled transcription–translation in vitro assay that upon expression in the presence of BH₄, wild-type PAH enzyme activity was enhanced. We then investigated the effect of BH₄ on PAH activity in transgenic mice that had a complete or partial deficiency in the endogenous cofactor biosynthesis. The rate of hepatic PAH enzyme activity increased significantly with BH₄ content without affecting gene expression or Pah-mRNA stability. These results indicate that BH₄ has a chaperon-like effect on PAH synthesis and/or is a protecting cofactor against enzyme auto-inactivation and degradation also in vivo. Our findings thus contribute to the understanding of the regulation of PAH by its cofactor BH₄ on an additional level and provide a molecular explanation for cofactor-responsive PKU.