Objectives—: Patients with mutations in the proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) gene have hypercholesterolemia and are at high risk of adverse cardiovascular events. We aimed to stably express the pathological human D374Y gain-of-function mutant form of PCSK9 ( PCSK9 ^DY ) in adult wild-type mice to generate a hyperlipidemic and proatherogenic animal model, achieved with a single systemic injection with adeno-associated virus (AAV). Approach and Results—: We constructed an AAV-based vector to support targeted transfer of the PCSK9 ^DY gene to liver. After injection with 3.5×10 ¹⁰ viral particles, mice in the C57BL/6J, 129/SvPasCrlf, or FVB/NCrl backgrounds developed long-term hyperlipidemia with a strong increase in serum low-density lipoprotein. Macroscopic and histological analysis showed atherosclerotic lesions in the aortas of AAV- PCSK9 ^DY mice fed a high-fat-diet. Advanced lesions in these high-fat-diet–fed mice also showed evidence of macrophage infiltration and fibrous cap formation. Hepatic AAV- PCSK9 ^DY infection did not result in liver damage or signs of immunologic response. We further tested the use of AAV- PCSK9 ^DY to study potential genetic interaction with the ApoE gene. Histological analysis of ApoE ^−/− AAV- PCSK9 ^DY mice showed a synergistic response to ApoE deficiency, with aortic lesions twice as extensive in ApoE ^−/− AAV- PCSK9 ^DY -transexpressing mice as in ApoE ^−/− AAV- Luc controls without altering serum cholesterol levels. Conclusions—: Single intravenous AAV- PCSK9 ^DY injection is a fast, easy, and cost-effective approach, resulting in rapid and long-term sustained hyperlipidemia and atherosclerosis. We demonstrate as a proof of concept the synergy between PCSK9 ^DY gain-of-function and ApoE deficiency. This methodology could allow testing of the genetic interaction of several mutations without the need for complex and time-consuming backcrosses.