Background: In this study, we aimed to investigate the effect of Ruanmailing oral liquid on atherosclerosis and transforming growth factor (TGF)-β1/SMAD4 signaling pathway in apolipoprotein E-knockout (ApoE−/−) mice induced by a high-fat diet. Materials and Methods: A total of 40 ApoE−/− mice were randomly divided into five groups: control group, model group, low-dose group, high-dose group, and Lipitor group. Mice fed with standard diet formed the control group. ApoE−/− mice exhibited high-fat diet-induced atherosclerotic phenotype. The other four groups were high-fat diet model groups, low- and high-dose Ruanmailing groups (1.75 and 4.55 mL/kg/day, respectively), and Lipitor group (3.0 mg/kg/day). After 12 weeks of administration, the levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), and high-density lipoprotein-cholesterol (HDL-C) were measured by blood sampling from the orbital vein of the mice, and the pathological changes in thoracic aorta due to atherosclerosis were observed by hematoxylin and eosin (H and E) staining. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the concentration of serum TGF-β1, and reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis were performed to detect the expression of SMAD4 and GATA2 in the thoracic aorta of mice in each group. Results: Compared with the high-fat model group, the level of serum lipids in the test group were reduced (P < 0.01 or P < 0.05) and the ratio of plaque area to luminal area (W/L) was significantly reduced (P < 0.05). The pathological examination indicated that the atherosclerotic lesions in the thoracic aorta of ApoE−/− mice were alleviated, and the high-dose Ruanmailing group had the most significant anti-atherosclerotic effect. Conclusion: Ruanmailing oral liquid exhibited an anti-atherosclerotic effect, and its mechanism may be related to the intervention of GATA2 in the TGF-β1/SMAD4 signaling pathway to reduce the differentiation and proliferation of arterial smooth muscle cells.