Abstract: To investigate the ameliorative effects and potential molecular mechanisms of Cistanche deserticola polysaccharides (CDPS) on metabolic dysfunction-associated fatty liver disease (MAFLD) induced by a high-fat and high-sugar diet, sixty male C57BL/6 mice were randomly divided into normal control, model, low-dose CDPS (100 mg/kg), medium-dose CDPS (200 mg/kg), high-dose CDPS (400 mg/kg), and Semaglutide (0.5 mg/kg) positive control groups. Overall efficacy was evaluated via serum biochemistry and liver pathological staining, while core hepatic targets and regulatory networks were systematically analyzed using combined transcriptomic sequencing and Western blot techniques. The results indicated that CDPS dose-dependently reduced body weight, liver index, and lipid levels in MAFLD mice. Furthermore, it significantly alleviated hepatic steatosis, inflammatory infiltration, and ballooning degeneration (P<0.05 or P<0.01), with the high-dose group showing efficacy comparable to Semaglutide. Transcriptomic analysis revealed that differentially expressed genes were mainly enriched in JAK-STAT, TNF, and cytokine-cytokine receptor interaction signaling pathways. Western blot assays confirmed that CDPS reversed the downregulation of TLR4, MYD88, STAT3, SOCS3, IL-10, SOD2, GPX4, and ITGB1, while inhibiting the upregulation of CCNB1, CDK1, PCNA, p53, α-SMA, COL1A1, CYP2E1, and MMP2 (P<0.05 or P<0.01). In conclusion, CDPS significantly ameliorates hepatic injury in MAFLD mice by synergistically regulating lipid metabolism, oxidative stress, inflammatory response, and fibrosis progression through multiple targets and pathways, providing experimental evidence for the prevention and treatment of metabolic fatty liver disease.