Adenosine receptors and extracellular adenosine have been demonstrated to modulate vascular smooth muscle cell (VSMC) proliferation and neointima formation. Adenosine kinase (ADK) is a major enzyme regulating intracellular adenosine levels, but is function in VSMC remains unclear. Here, we investigated the role of ADK in vascular injury-induced smooth muscle proliferation and delineated the mechanisms underlying its action. We found that ADK expression was higher in the neointima of injured vessels and in PDGF-treated VSMCs. Genetic and pharmacological inhibition of ADK was enough to attenuate arterial injury-induced neointima formation due to inhibition of VSMC proliferation. Mechanistically, using infinium methylation assays and bisulfite sequencing, we showed that ADK metabolized the intracellular adenosine and potentiated the transmethylation pathway, then induced the aberrant DNA hypermethylation. Pharmacological inhibition of aberrant DNA hypermethylation increased KLF4 expression and suppressed VSMC proliferation as well as the neointima formation. Importantly, in human femoral arteries, we observed increased ADK expression and DNA hypermethylation as well as decreased KLF4 expression in neointimal VSMCs of stenotic vessels suggesting that our findings in mice are relevant for human disease and may hold translational significance. Our study unravels a novel mechanism by which ADK promotes VSMC proliferation via inducing aberrant DNA hypermethylation, thereby downregulating KLF4 expression and promoting neointima formation. These findings advance the possibility of targeting ADK as an epigenetic modulator to combat vascular injury. Abnormal proliferation of vascular smooth muscle cell (VSMC) is key to abundant occlusive vascular diseases in humans, such as atherosclerosis and intimal hyperplasia associated with restenosis. Adenosine has been shown to combat abnormal smooth muscle proliferation. Here, we demonstrate that increased catabolism of adenosine by adenosine kinase (ADK) promotes abnormal VSMC proliferation. The pathological ADK overexpression in both mice and humans with vascular disease promotes VSMC proliferation via inducing aberrant DNA hypermethylation and KLF4 downregulation. Our study suggests that pharmacological augmentation of endogenous adenosine by targeting ADK represents a promising therapeutic strategy for occlusive vascular diseases. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions please email: journals.permissions@oup.com.