To investigate the effect of trimethylamine oxide (TMAO)-driven PERK/FOXO1 axis on HDL metabolism in palmitic acid (PA)-induced human hepatocellular carcinoma (HepG2) cells. HepG2 cells were cultured in vitro, and the inhibition rates of PA, TMAO, and DMB (3,3-dimethyl-1-butanol, a TMAO inhibitor) were detected by CCK-8, respectively, and the final concentrations were taken for the subsequent experiments. Changes in the cellular TG, TC, HDL-C, and LDL-C contents were detected by the biochemical kit method, and cellular lipid deposition was detected by the Oil Red O staining method. Western Blot method was used to detect cellular PERK, FOXO1 SR-BI, HL, APOA-1 protein expression. The final concentration of 0.2 mmol/L PA, 10 μmol/L DMB, and 0.4 mmol/L TMAO were selected for the final experiment, and were randomly divided into 6 groups: Control group, DAB control group (10 μmol/L DAB, TMAO inhibitor), PA-induced group (0.2 mmol/L palmitic acid), PA + DAB control group (0.2 mmol/L PA + 10 μmol/L DAB), TMAO group (0.2 mmol/LPA + 0.4 mmol/L TMAO), TMAO + DAB group (0.2 mmol/L PA +0.4mmol/L TMAO + 10 μmol/L DAB). The results show that compared with the Control group, the PA and TMAO groups show significantly higher TG, TC, and LDL-C content (P <0.01), significantly lower HDL-C content (P < 0.01), significant cellular lipid deposition, elevated intracellular p-PERK and FOXO1 protein expression (P < 0.05, P < 0.01), and SR-BI, HL, APOA-1 protein expression is significantly reduced (P <0.01). Compared with the PA group, the changes of the above indexes in the TMAO group are further aggravated (P <0.05, P < 0.01). Compared with the TMAO group, the changes of the indexes in the TMAO + DMB group are significantly improved after the administration of inhibitors (P <0.05, P < 0.01). It can be seen that TMAO can achieve the role of regulating downstream HDL metabolism by driving the PERK/FOXO1 axis.