The primary purpose of this study is to assess the safety and tolerability of AG-181 in subjects with Phenylketonuria (PKU).

Key Inclusion Criteria:

• Diagnosis of PKU, defined as documented presence of 2 mutant alleles in the phenylalanine hydroxylase (PAH) gene, of which at least 1 is the R408W mutation, as determined during Screening per the genotyping performed by the study central genotyping laboratory.
• At least 1 plasma Phe concentration greater than (>) 600 micromoles per liter (μmol/L) in the 52 weeks before providing informed consent.
• Average concentration of plasma Phe > 600 μmol/L in Phe samples taken during Screening, with no individual assessment below 360 μmol/L. Any Phe samples taken after Day -20 will not be included.
• Body mass index (BMI) greater than or equal to (≥) 18.0 kilograms per meter square (kg/m^2) to lesser than or equal to (≤) 35.0 kg/m^2 and weight ≥ 50 kilograms (kg) at any time during the Screening Period.
• Documented approval from a dietitian confirming that the subject can maintain their diet consistent in protein and Phe intake throughout the study as outlined in the Diet Manual.

Key Exclusion Criteria:

• Prior exposure to AG-181.
• Receiving inhibitors of P-glycoprotein (P-gp) that have not been stopped for ≥ 5 days or a timeframe equivalent to 5 half-lives (whichever is longer) before administration of the first dose of study drug.
• Receiving products that are strong inhibitors or strong inducers of cytochrome P450 CYP1A2, CYP2C8, or CYP3A that have not been stopped for ≥ 28 days before administration of the first dose of study drug.
• Receiving treatment with an acid-reducing agent, including but not limited to proton pump inhibitors and H2 blockers. Short-acting acid-reducing agents such as calcium carbonate are permitted.
• Any preexisting condition that could (in the opinion of the Investigator) interfere with gastrointestinal anatomy or motility that may disrupt the absorption, metabolism, and/or excretion of the study drug.
• Any preexisting condition that could (in the opinion of the Investigator) interfere with hepatic or renal function that may disrupt the absorption, metabolism, and/or excretion of the study drug.
• Inability to tolerate oral medication.
• Unwillingness to washout from tetrahydrobiopterin (BH4) supplementation (eg, sapropterin dihydrochloride, Kuvan), pegvaliase-pqpz (Palynziq), or any other PKU therapy by Day -30 during Screening.