Aim The aim of the present study is to determine the function of signal transducer and activator of transcription 4 (STAT4) signal in macrophage differentiationand foam cell formation during the development of atherosclerosis. Methods STAT4 knockout (STAT4－/－) mice were crossed with Apolipoprotein E knockout mice (ApoE－/－)to establish a novel atherosclerosis related mice model- APOE/STAT4 double knockout (DKO) mice. WT,STAT4－/－,ApoE－/－ and DKOmice (3-month-old adult mice) were challenged with high-fat diet for 12 weeks. The extent of atherosclerosis was determined by oil-red staining and HE staining. Changes in subsets of immune cells were evaluated by flow cytometry. Macrophage differentiation was induced from the CD11b⁺ myeloid cells directly isolated from the bone marrow of transgenic mice with macrophage colony stimulating factor (M-CSF) incubation. Realtime polymerase chain reaction (RT-PCR) and Westernblot were applied to detect the expression levels of related genes and protein. Results The expression of STAT4 was identified in CD11b⁺ myeloid cells in atherosclerotic plaque. Genetic deletion of STAT4 significantly exacerbated atherosclerosis in ApoE－/－ mice as evidenced by significant increases of oilred O-positive lipid-rich lesion, vascular inflammation, and necrotic core lesion in atherosclerotic plaques of DKO mice compared to ApoE－/－mice. The accelerated atherosclerotic process is associated with abnormal mobilization and differentiation of macrophages from CD11b⁺Gr-1⁺ immature myeloid cells and with the enhanced formation of macrophages-derived foam cells in DKO mice. Furthermore,the mechanism studies revealed that the disruption of STAT4 signal leads to the inhibition of phosphatidylinositol-3 kinase(PI3K)/serine-threonine kinases (Akt)/nuclear factor kappa B (NF-κB)/miR-9 signaling pathway and consequently up-regulates the expression of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1), an enzyme that esterifies cholesterol and promotes its storage in macrophages and foam cells. Conclusion The results suggest that the STAT4 signal plays important roles in both immune responses and cholesterol metabolism during the differentiation of macrophages. Targeting STAT4 related signals may develop a novel pharmacotherapeutic target for the treatment of atherosclerotic diseases.