Endoplasmic reticulum (ER) stress-mediated apoptosis is a key feature of hepatocyte cytotoxicity by saturated free fatty acids (FFA). This lipoapoptosis is dependent, in part, on the transcriptional upregulation of the BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Although the activator protein (AP)-1 complex facilitates PUMA expression by saturated FFA, the transcription factor CAAT/enhancer binding homologous protein (CHOP) is also induced by ER stress and promotes apoptosis. To integrate the role of these two transcription factors in ER stress-induced apoptosis, we examined the relative contribution of CHOP and AP-1 in mediating PUMA induction by saturated FFA. Our results demonstrate that short-hairpin RNA-targeted knockdown of CHOP attenuates palmitate-induced apoptosis in Huh-7 cells. Loss of CHOP induction also reduced the increase in PUMA mRNA and protein levels as well as Bax activation by palmitate. No functional CHOP binding sites were identified in the PUMA promoter sequence. Rather, we observed that CHOP physically interacts with the AP-1 complex protein c-Jun upon palmitate treatment, and a CHOP:phosphorylated c-Jun heteromeric complex binds to the AP-1 consensus binding sequence within the PUMA promoter region. Finally, loss of function studies suggest that both transcription factors are necessary for maximal PUMA induction. Collectively, these data suggest that CHOP and AP-1 cooperatively mediate PUMA induction during hepatocyte lipoapoptosis.