Phenyl butyrate (PB) has been proved to decrease pyruvate dehydrogenase (PDH) phosphorylation level and increase PDH activity by inhibiting pyruvate dehydrogenase kinase 1 (PDK1) in fibroblast cells, PDH deficiency zebrafish and wild type mice. PB has also shown efficacy in many cancers and so far, all of its anti-tumor activity has been attributed to the histone deacetylase (HDAC) inhibition. As PDK1/PDH controls the critical switch between oxidative phosphorylation and glycolysis in cancer cells, PDK1 is a key target in tumor metabolism for anti-cancer treatment. We hypothesize that the therapeutic effects of PB in cancers might also depend on suppressing PDK1 and promoting PDH activity, in addition to its proposed role as HDAC inhibitor. We showed that PB directly inhibited the kinase activity of PDK1 and increased the activity of PDH in an enzyme assay. In several different cancer cell lines, PB reduced the phosphorylation level of PDH, increased the mitochondrial respiration, decreased glycolysis in cytoplasm, reversed mitochondrial hyperpolarization, activated several proteins in apoptotic signaling pathway and then induced the apoptosis of cells. In summary, this is the first study indicated that PB could exert its anti-cancer effects through inhibiting PDK1, altering the mitochondrial bioenergetics and inducing apoptosis.