The adeno-associated virus (AAV) has many safety features that favor its use in the treatment of arteriosclerosis; however, the conventional, adeno-associated virus (AAV) mediated single-gene delivery is inefficient for arteriosclerosis. This has been attributed that the incidence of atherosclerosis is caused by a variety of genetic defects but not a particular gene. To overcome this, double-gene delivery was evaluated for the treatment of atherosclerosis. Four experimental groups were administered the following AAV vector constructs: rAAV-apoAⅠ-IRES-SR-BⅠ, rAAV-apoAⅠ-GFP, rAAV-IRES-GFP, and PBS. ApoAⅠ and SR-BⅠ gene expression was detected using RT-PCR. The apoAⅠ and SR-BⅠ protein expression was determined by Western blotting and ELISA. Diet-induced hypercholesterolemia and atherosclerosis in rats was adopted and rAAV was administered through the tail vein injection. HepG2 cells were cultured and infected with the three viral vectors. The apoAⅠ and SR-BⅠ secreted from HepG2 cells in the AAV- apoAⅠ/SR-BⅠ group enhanced cholesterol efflux and resulted in a stronger RCT ability, respectively. In the rats' model with diet-induced hypercholesterolemia and atherosclerosis, GFP expression could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, plasma total cholesterol and LDL-cholesterol concentrations were significantly reduced (P < 0.05) compared to control for rAAV-IRES-GFP (AAV-GFP) treated group. No effect on HDL-cholesterol concentrations occurred. Ultrasound determined intima-media thickness also has been significantly reduced compared to control. Serum hs-CRP and SOD levels increased significantly (P < 0.01). Serum MDA levels decreased significantly. Gene mRNA expression was detected in atherosclerosis rats' model. The results show that rAAV-hapoAⅠ-IRES-hSR-BⅠ vector can anti-inflammatory, reduce atherosclerotic macrophage content and increases lesion stability of pre-existing plaques through quenching of NF-κB activity and reducing plasma cholesterol. Simultaneous over-expression of apoAⅠ and SR-BⅠ by AAV-mediated gene transfer may have a favorable effect on diet-induced hypercholesterolemia and arteriosclerosis in rats. These results may provide a new method for gene therapy of arteriosclerosis.