Free fatty acids (FFA) induce hepatocyte lipoapoptosis by a c-Jun N-terminal kinase (JNK)-dependent mechanism. However, the cellular processes by which JNK engages the core apoptotic machinery during lipotoxicity, especially activation of BH3-only proteins, remain incompletely understood. Thus, our aim was to determine whether JNK mediates induction of BH3-only proteins during hepatocyte lipoapoptosis. The saturated FFA palmitate, but not the monounsaturated FFA oleate, induces an increase in PUMA mRNA and protein levels. Palmitate induction of PUMA was JNK1-dependent in primary murine hepatocytes. Palmitate-mediated PUMA expression was inhibited by a dominant negative c-Jun, and direct binding of a phosphorylated c-Jun containing the activator protein 1 complex to the PUMA promoter was identified by electrophoretic mobility shift assay and a chromatin immunoprecipitation assay. Short hairpin RNA-targeted knockdown of PUMA attenuated Bax activation, caspase 3/7 activity, and cell death. Similarly, the genetic deficiency of Puma rendered murine hepatocytes resistant to lipoapoptosis. PUMA expression was also increased in liver biopsy specimens from patients with non-alcoholic steatohepatitis as compared with patients with simple steatosis or controls. Collectively, the data implicate JNK1-dependent PUMA expression as a mechanism contributing to hepatocyte lipoapoptosis.