Abstract Propionic acidemia (PA) is a rare metabolic disease due to PCCA or PCCB mutations. PA could develop various complications even with dietary restriction. While gene therapy holds promise, it remains hampered by challenges. Hence, there is a pressing need to develop novel, effective, and systemic therapy for PA. PCC deficiency leads to propionyl-CoA accumulation, depleting free CoA and inhibiting acetyl-CoA synthesis. This reduces the C2/C3 ratio from 11 to 0.08 in PA mouse model. Elevated propionyl-CoA disrupts acetyl-CoA- involved pathways, impeding energy metabolism (propionyl-CoA, methylcitrate), causing hyperammonemia (propionylglutamate), and affecting histone acetylation and gene transcription. Restoring the acetyl-CoA level, particularly the C2/C3 ratio, is crucial to prevent metabolic alterations and subsequent propionyl-CoA-driven pathology. Our preliminary data demonstrate the therapeutic efficiency of acetate supplementation, which improves the C2/C3 ratio and further overall survival of Pcca-/-(A138T) mice. Triacetin, an FDA-approved food additive, is a glycerol and acetic acid triester that undergoes rapid metabolism into glycerol and acetate upon absorption. This makes it a secure and effective means of supplementing acetate. Therefore, we hypothesize that balancing the acetyl-CoA/propionyl-CoA ratio via triacetin supplementation offers therapeutic potential for PA, bolstering acetyl-CoA synthesis and competitively impeding propionate metabolism. To test our hypothesis, we have formulated the following two specific aims. Aim 1. To evaluate the efficacy of triacetin in rectifying metabolic abnormalities, cellular phenotypes and functions in human PA cells and its pharmacokinetics and pharmacodynamics (PK/PD) in vitro. We will utilize human PA fibroblasts and PA hepatocyte (PCCAA138T-HepG2) to examine whether triacetin can mitigate propionyl-CoA metabolism, cellular phenotypes and function in vitro. PK/PD of triacetin will be examined to establish correlations between its efficacy and these parameters. Aim 2. To assess the metabolic effects, therapeutic efficacy, and PK/PD of triacetin in Pcca-/-(A138T) mice in vivo. To thoroughly validate the therapeutic effectiveness of triacetin, we will conduct an in vivo study involving PK/PD analysis in Pcca-/-(A138T) mice. We will assess the metabolic impact of triacetin supplementation by examining parameters such as the C2/C3 ratio and the levels of propionylcarnitine, methylcitrate, and ammonium in the plasma and various tissues of Pcca-/-(A138T) mice. Additionally, we will perform tissue histological evaluations and monitor overall survival to gain valuable insights into the therapeutic potential of triacetin.