Background: Migration of human aortic smooth muscle cells (HASMCs) contributes to the pathogenesis of atherosclerosis. This study aims to functionally characterize long noncoding RNA TPRG1-AS1 (tumor protein p63 regulated 1, antisense 1) in HASMCs and reveal the underlying mechanism of TPRG1-AS1 in HASMCs migration, neointima formation, and subsequent atherosclerosis. Methods: The expression of TPRG1-AS1 in atherosclerotic plaques was verified a series of in silico analysis and quantitative real-time polymerase chain reaction analysis. Northern blot, rapid amplification of cDNA ends and Sanger sequencing were used to determine its full length. In vitro transcription-translation assay was used to investigate the protein-coding capacity of TPRG1-AS1. RNA fluorescent in situ hybridization was used to confirm its subcellular localization. Loss- and gain-of-function studies were used to investigate the function of TPRG1-AS1. Furthermore, the effect of TPRG1-AS1 on the pathological response was evaluated in carotid balloon injury model, wire injury model, and atherosclerosis model, respectively. Results: TPRG1-AS1 was significantly increased in atherosclerotic plaques. TPRG1-AS1 did not encode any proteins and its full length was 1279nt, which was bona fide a long noncoding RNA. TPRG1-AS1 was mainly localized in cytoplasmic and perinuclear regions in HASMCs. TPRG1-AS1 directly interacted with MYH9 (myosin heavy chain 9) protein in HASMCs, promoted MYH9 protein degradation through the proteasome pathway, hindered F-actin stress fiber formation, and finally inhibited HASMCs migration. Vascular smooth muscle cell-specific transgenic overexpression of TPRG1-AS1 significantly reduced neointima formation, and attenuated atherosclerosis in apolipoprotein E knockout ( Apoe ^−/− ) mice. Conclusions: This study demonstrated that TPRG1-AS1 inhibited HASMCs migration through interacting with MYH9 protein and consequently suppressed neointima formation and atherosclerosis.