Background: Most cardiovascular diseases are age-related, accompanied by cell senescence in the cardiovascular system. Ionizing radiation (IR) has been reported to be involved in the dysfunction of endothelial cells (ECs) and smooth muscle cells. However, the specific mechanism is poorly defined. This study explores a possible mechanism of IR-induced cell senescence. Methods: Human umbilical vein ECs (HUVECs) and human aortic smooth muscle cells (HASMCs) were irradiated with 20 Gy X-rays for 6 days to induce cell senescence. Cell transfection was then used to overexpress or delete myeloid zinc finger 1 (MZF1) and neogenin 1 (NEO1) in HUVECs and HASMCs, along with the determination of their expressions. Cell viability, senescence associated-beta-galactosidase (SA-beta-gal) levels, and expressions of senescence markers were determined to assess cell senescence. Results: In HUVECs and HASMCs, irradiation upregulated MZF1 and NEO1 expressions, inhibited cell viability, and elevated SA-beta-gal level. However, MZF1 overexpression enhanced and MZF1 deletion reversed the effect of irradiation. Additionally, MZF1 deletion blocked NEO1 expression in irradiated HUVECs and HASMCs. NEO1 overexpression repressed cell viability, accelerated the SA-beta-gal increase and upregulated expressions of cyclin dependent kinase inhibitor 2A/1A (p16/21), p53 and high mobility group AT-hook 1 (Hmga1) in irradiated HUVECs and HASMCs, which was counteracted by MZF1 deletion. Also, NEO1 overexpression reversed the effects of MZF1 deletion in irradiated HUVECs and HASMCs. Conclusion: MZF1 and NEO1 mediate IR-induced senescence of cardiovascular cells. This may provide a window for exploiting new targets in the therapy of age-related cardiovascular diseases.