Objective To investigate the effect of TMAO, a metabolite of intestinal flora, on hepatic lipid metabolism in rats with splenic deficiency and hyperlipidemia, and to further explore the possible mechanism of Xiangsha Liujunzi Decoction in the treatment of hepatic lipid metabolism disorder. Methods SD rats were divided a blank control group (C group), blank control + TMAO inhibitor DMB group (C + D group), spleen deficiency hyperlipidemia group (PG group), spleen deficiency hyperlipidemia + DMB group (PG + D group), and spleen deficiency hyperlipidemia + Xiangsha Liujunzi Decoction group (PG + XS group). Except C group and C + D group, the other groups were used to establish a spleen deficiency hyperlipidemia model (12 weeks of modeling) by combining excessive fatigue and a high fat diet. After model establishment, C + D group and PG + D group were administered 1%DMB in drinking water every day, and the PG + XS group was administered Xiangsha Liujunzi Decoction (11. 34 g crude drug/ kg) every day. The other groups were administered the same amount of normal saline. Blood lipid levels were measured by an automatic biochemical method after 4 weeks of intragastric administration. Morphological changes of the liver were observed by HE staining. Lipid deposition in the liver was observed by oil red O staining. Liver FFA, TG, and TC were measured by ELISA. Plasma TMAO content was measured by LC-MS. Relative mRNA expression levels of PERK, FOXO1, SREBP-2, ABCA1, and miR-33 in the liver were measured by qRT-PCR. SREBP-2 and ABCA1 contents in the liver were measured by Western Blot. Results Serum contents of TC, TG, and LDL-C in the PG group were significantly higher than those in the C group, and HDL-C was content was significantly lower than that in the C group. FFA, TG, and TC contents in liver tissue of PG rats were significantly increased compared with those the C group. Compared with the C group, lipid deposition in the liver was aggravated and vacuoles were increased significantly. There was no difference in the above indexes between group C + D group and C group. Compared with the PG group, PG + D group and PG + XS group had significantly reduced serum contents of TC, TG, and LDL-C, increased HDL-C content, reduced FFA, TG, and TC contents in liver tissue, alleviated lipid deposition in liver tissue, and reduced liver vacuoles. No significant difference was observed between PG + D group and PG + XS group. Compared with the C group, the plasma TMAO content of the PG group was significantly increased, mRNA expression PERK, FOXO1, and miR-33a in the liver was significantly increased, and liver SREBP-2 and ABCA1 mRNA and protein expression was significantly decreased. No difference in the above indexes was observed between C + D and C groups. Compared with the PG group, PG + D group and PG + XS group had significantly reduced plasma TMAO content, decreased mRNA expression of PERK, FOXO1, and miR-33a in the liver, and increased SREBP-2 and ABCA1 mRNA and protein expression in the liver. No significant difference was observed between PG + D group and PG + XS group. Conclusions TMAO may regulate the SREBP-2/ miR-33a/ ABCA1 signaling pathway through the PERK/ FOXO1 axis to cause liver lipid metabolism disorder in rats, and Xiangsha Liujunzi Decoction may inhibit liver lipid metabolism disorder by reducing TMAO content.