Hepatic lipotoxicity, resulting from excessive lipid accumulation in hepatocytes, plays a central role in the pathogenesis of various metabolic liver diseases. Despite recent progress, the precise mechanisms remain incompletely understood. Using excessive exposure to palmitate in hepatocytes as our primary experimental model and mice studies, we aimed to uncover the mechanisms behind hepatic lipotoxicity, thereby developing potential treatments. Our data reveal for the first time that exposure to palmitate leads to downregulated expression of poly (ADP-ribose) polymerase 1 (PARP-1) in hepatocytes, inhibiting its enzymatic activity. Whereas inhibiting PARP-1 worsens palmitate-induced hepatotoxicity, preventing PARP-1 suppression, using nicotinamide adenine dinucleotide (NAD(+)) precursors, nicotinamide N-methyltransferase (NNMT) inhibitors, or a poly(ADP-ribose) glycohydrolase (PARG) inhibitor, prevents it. Moreover, we uncover that PARP-1 suppression contributes to palmitate-triggered mechanistic target of rapamycin complex 1 (mTORC1) activation, which has been previously reported by us to contribute to palmitate-induced hepatocyte cell death. Furthermore, our results identify p300 as a downstream target of mTORC1 activation upon palmitate exposure. Importantly, p300 inhibition via either pharmacological or genetic approaches protects against palmitate hepatotoxicity. In addition, we provide evidence that the toll-like receptor 4 (TLR4)-nuclear factor κB (NF-κB) pathway activation in response to palmitate plays a mechanistic role in mediating palmitate-induced PARP-1 downregulation in that both TLR4 antagonist and NF-κB inhibitors prevent palmitate-induced PARP-1 reduction and protect against hepatocyte cell death. In conclusion, our study presents new evidence that the PARP-1-mTORC1-p300 pathway serves as a novel molecular mechanism underlying palmitate-induced hepatic lipotoxicity. Targeting the PARP-1 pathway by increasing cellular NAD(+) availability either through its precursor supplementation or by inhibiting its degradation represents a promising therapeutic approach for treating hepatic lipotoxicity.NEW & NOTEWORTHY This study explores the mechanisms of palmitate-induced hepatotoxicity, highlighting the role of PARP-1 downregulation in triggering the mTORC1-p300 pathway and resultant hepatocyte cell death. It further reveals that enhancing cellular NAD(+) levels through either precursor supplementation or NNMT inhibitors prevents lipotoxicity by restoring PARP-1 activity. Finally, the study identifies that the TLR4-NF-κB activation mediates palmitate-induced PARP-1 suppression and offers potential therapeutic insights for metabolic liver diseases caused by lipotoxicity.