The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play vital roles in neointimal hyperplasia and vascular restenosis. In the present study, we aimed to investigate the function and mechanism of octamer-binding transcription factor 4 (OCT4, a key transcription factor for maintaining stem cells in de-differentiated state) on neointima formation in response to vascular injury. Quantitative reverse-transcription polymerase chain reaction and western blot results displayed a significant increase of OCT4 levels in injured carotid arteries. Immunohistochemistry and immunofluorescence assays confirmed that the increased OCT4 expression was primarily localized in α-SMA-positive VSMCs from neointima, and colocalized with PCNA in the nuclei of VSMCs. Adenovirus-mediated OCT4 overexpression in injured carotid arteries exacerbated intimal thickening, while OCT4 knockdown significantly inhibited intimal thickening. In-vitro experiments confirmed that the increased OCT4 expression in VMSCs could be induced by platelet-derived growth factor-BB (PDGF-BB) in a time-dependent manner. Overexpression of OCT4 greatly promoted VSMCs proliferation and migration, while OCT4 knockdown significantly retarded the PDGF-BB-induced excessive proliferation and migration of VSMCs. Bioinformatics analysis, dual-luciferase reporter assay, and chromatin immunoprecipitation assay confirmed that OCT4 could upregulate matrix metalloproteinases 2 (MMP2) expression through promoting its transcription. Moreover, knockdown of MMP2 significantly attenuated OCT4-mediated VSMCs proliferation and migration. These results indicated that OCT4 facilitated neointimal formation in response to vascular injury by MMP2-mediated VSMCs proliferation and migration, and targeting OCT4 in VSMCs might be a novel therapeutic strategy for vascular restenosis.