Saturated free fatty acids (FFA) induce hepatocyte lipoapoptosis, a key mediator of liver injury in nonalcoholic fatty liver disease (NAFLD). Lipoapoptosis involves the upregulation of the BH3-only protein PUMA, a potent pro-apoptotic protein. Given that dysregulation of hepatic microRNA expression has been observed in NAFLD, we examined the role of miRNA in regulating PUMA expression during lipotoxicity. By in silico analysis, we identified two putative binding sites for miR-296-5p within the 3' untranslated region (UTR) of PUMA mRNA. Enforced miR-296-5p levels efficiently reduced PUMA protein expression in Huh-7 cells, while antagonism of miR-296-5p function increased PUMA cellular levels. Reporter gene assays identified PUMA 3'UTR as a direct target of miR-296-5p. The saturated FFA, palmitate, repressed miR-296-5p expression; and Huh-7 cells were sensitized to palmitate-induced lipotoxicity by antagonism of miR-296-5p function using a targeted locked nucleic acid (LNA). Finally, miR-296-5p was reduced in liver samples from nonalcoholic steatohepatitis (NASH) patients compared with patients with simple steatosis (SS) or controls. Also miR-296-5p levels inversely varied with PUMA mRNA levels in human liver specimens. Our results implicate miR-296-5p in the regulation of PUMA expression during hepatic lipoapoptosis. We speculate that enhancement of miR-296-5p expression may represent a novel approach to minimize apoptotic damage in human fatty liver diseases.