Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2ΔRnH) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2ΔRnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pahenu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 1014 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.