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Background: The modulation of vascular smooth muscle cells (VSMC) phenotypes is a highly important process in the formation of neointima and in resolution of inflammation after vascular injury. Underlying genetic mechanisms that control the phenotypic switch of VSMC in disease are not completely understood. On this basis, the phenotypic regulation of VSMC in restenosis development was examined in the mouse carotid artery injury model. Methods and Results: Wire-induced injury, an established mouse-model to study restenosis, was applied to induce vascular injury in C57BL/6 mice. Time-resolved single-cell RNA-sequencing (scRNAseq) and massive-analysis-of-cDNA-ends (MACE)-RNAseq of neointima obtained by laser capture microdissection were performed to detect genetic changes in neointima development. Single-cell RNA-sequencing of C57BL/6 mice at different time points after wire-induced injury revealed an expanding, highly proliferative activated VSMC cluster, which exhibited low expression of SMC marker genes. Analysis for differentially regulated genes (DEG) and pseudotime clustering identified a unique marker gene signature, including high expression of TIMP Metallopeptidase Inhibitor 1 (Timp1), which was identified as main ordering gene of the modulation processes′ pseudo temporal states. MACEseq confirmed upregulation of Timp1 selectively in neointima forming VSMCs. Integration with atherosclerosis scRNAseq data indicated differences in gene signatures of activated VSMC populations. Transcription factor analysis of modulated VSMC identified BACH1, a factor involved in reactive oxidative stress response and proliferation of SMCs, as potential regulatory target for re-differentiation of VSMCs after vascular injury. Conclusion: ScRNAseq identified a unique cell cluster responsible for neointima formation. Targeting the transcription factors driving the cluster (like BACH1) can be exploited to combat neointima development.