Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common disorder leading to lactic acidemia. Phosphorylation of specific serine residues of the E1-alpha subunit of the PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We recently found that phenylbutyrate prevents phosphorylation of the E1-alpha subunit of the branched-chain ketoacid dehydrogenase complex (BCKDC) and reduces plasma concentrations of neurotoxic branched chain amino acids in patients with maple syrup urine disease (MSUD), due to the deficiency of BCKDC. We hypothesized that, similarly to BCKDC, phenylbutyrate enhances PDHC enzymatic activity by increasing the portion of unphosphorylated enzyme. To test this hypothesis, we treated wild-type human fibroblasts at different concentrations of phenylbutyrate and found that it reduces the levels of phosphorylated E1-alpha as compared to untreated cells. To investigate the effect of phenylbutyrate in vivo, we administered phenylbutyrate to C57B6 wild-type mice and we detected a significant increase in Pdhc enzyme activity and a reduction of phosphorylated E1-alpha subunit in brains and muscles as compared to saline treated mice. Being a drug already approved for human use, phenylbutyrate has great potential for increasing the residual enzymatic activity of PDHC and to improve the clinical phenotype of PDHC deficiency.