The mechanism by which Xiangsha Liujunzi Decoction targets the trimethylamine N-oxide (TMAO)-mediated PERK/FOXO1 axis to regulate downstream high-density lipoprotein (HDL) metabolism was investigated in rats with spleen dysfunction and impaired lipid transport and transformation. Thirty-five Sprague-Dawley (SD) rats were divided into five groups: normal control group (Z group), TMAO inhibitor (DMB) group (D group), spleen dysfunction with impaired lipid transport and transformation group (PSJY group), model plus DMB intervention group (PSJY+D group), and model plus Xiangsha Liujunzi Decoction intervention group (PSJY+XS group). Except for the Z and D groups, all other groups were fed with a high-fat diet combined with overwork for 12 weeks to establish the model. From the 13th week onwards, 1% DMB was added to the drinking water of the D and PSJY+D groups. The PSJY+XS group was administered Xiangsha Liujunzi Decoction[11.34 g crude drug/(kg/d)] by gavage for 4 weeks. The experimental results show that, compared with the Z group, significant hepatic lipid deposition is observed in the PSJY group. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and apolipoprotein B (ApoB) are significantly elevated in the PSJY group (P<0.01). Serum high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (ApoA-I) are significantly decreased (P<0.01). Plasma TMAO level is increased (P<0.01). Protein expressions of phosphorylated protein kinase RNA-like endoplasmic reticulum kinase(p-PERK) and forkhead box O1(FOXO1) in liver tissue are increased (P<0.01). mRNA and protein expressions of scavenger receptor class B type I (SR-BI), ApoA-I, and hepatic lipase (HL) are decreased (P<0.01). No significant changes in most indicators are found in the DMB intervention group, except for decreased TMAO and downregulated SR-BI mRNA expression. Compared with the PSJY group, fewer hepatic lipid droplets are observed in the PSJY+D group. Serum TG and ApoB are decreased (P<0.01), and serum ApoA-I is increased (P<0.05). TMAO level is significantly decreased (P<0.01). Protein expressions of SR-BI, ApoA-I, and HL are increased (P<0.05 or P<0.01). In the PSJY+XS group, hepatic lipid deposition is alleviated. Serum TG, LDL-C, and ApoB are decreased (P<0.01). Serum HDL-C and ApoA-I are increased (P<0.01 for HDL-C, P<0.05 for ApoA-I). TMAO level is decreased (P<0.01). Protein expressions of p-PERK/PERK and FOXO1 are decreased (P<0.05). mRNA and protein expressions of SR-BI, ApoA-I, and HL are significantly increased (P<0.05 or P<0.01). Under the condition of spleen dysfunction with impaired lipid transport and transformation, alterations in the TMAO/PERK/FOXO1 axis are shown to lead to downstream HDL metabolic disorder. HDL metabolic disorder in this condition is alleviated by Xiangsha Liujunzi Decoction through targeting the TMAO-mediated PERK/FOXO1 axis.