Abstract: Objective To evaluate the effect of palmitic acid (PA) on oxidative stress and activation of inflammasomes in hepatocytes. Methods To test the dose-dependent effect of PA on normal murine hepatocytes AML12, the cells were treated with 0, 0.15, 0.25 and 0.4 mmol/L of palmitic acid (PA). The cells were also divided into blank control group, 0.25 mmol/L PA group and 0.25 mmol/L PA+ N-acetylcysteine (NAC) group to examine the effect of reactive oxygen species (ROS) on the activation of inflammasomes. After 24 h of treatment, lipid accumulation, total ROS, mitochondrial ROS, expression and localization of NOX4, and expressions of inflammasomes and IL-1β were detected in the hepatocytes. Results Compared with the control cells, PA treatment of the cells significantly increased cytoplasmic lipid accumulation, concentrations of total ROS (12 463.09 ± 2.72 vs 6691.23 ± 2.45, P=0.00) and mitochondrial ROS (64.98 ± 0.94 vs 45.04 ± 0.92, P=0.00), and the expressions of NOX4, NLRP3, ASC, caspase-1, and IL-1β (1603.52±1.32 vs 2629.33±2.57, P=0.00). The mitochondria and NOX4 were found to be co-localized in the cytoplasm. NAC obviously reduced cellular ROS level stimulated by PA (7782.15±2.87 vs 5445.6±1.17, P= 0.00) and suppressed the expressions of NLRP3, ASC and caspase-1. Conclusion PA treatment can stimulate lipid accumulation in hepatocytes and induce oxidative stress through NOX4 and mitochondria pathway to activate inflammasomes and stimulate the secretion of IL-1β.