Death-associated protein kinase 3 (DAPK3) also known as zipper-interacting kinase is a serine/threonine kinase that mainly regulates cell death and smooth muscle contraction. We have previously found that protein expression of DAPK3 increases in the mesenteric artery from spontaneously hypertensive rats (SHRs) and that DAPK3 mediates the development of hypertension in SHRs partly through promoting reactive oxygen species-dependent vascular inflammation. However, it remains to be clarified how DAPK3 controls smooth muscle cell (SMC) proliferation and migration, which are also important processes for hypertension development. We, therefore, sought to investigate whether DAPK3 affects SMC proliferation and migration. siRNA against DAPK3 significantly inhibited platelet-derived growth factor (PDGF)-BB-induced SMC proliferation and migration as determined by bromodeoxyuridine (BrdU) incorporation and a cell counting assay as well as a Boyden chamber assay respectively. DAPK3 siRNA or a pharmacological inhibitor of DAPK3 inhibited PDGF-BB-induced lamellipodia formation as determined by rhodamine-phalloidin staining. DAPK3 siRNA or the DAPK inhibitor significantly reduced PDGF-BB-induced activation of p38 and heat-shock protein 27 (HSP27) as determined by Western blotting. In ex vivo studies, PDGF-BB-induced SMC out-growth was significantly inhibited by the DAPK inhibitor. In vivo, the DAPK inhibitor significantly prevented carotid neointimal hyperplasia in a mouse ligation model. The present results, for the first time, revealed that DAPK3 mediates PDGF-BB-induced SMC proliferation and migration through activation of p38/HSP27 signals, which may lead to vascular structural remodelling including neointimal hyperplasia. The present study suggests DAPK3 as a novel pharmaceutical target for the prevention of hypertensive cardiovascular diseases.