Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genomic region contains the CDKN2A and CDKN2B genes which encode the cell cycle regulators p16^INK4a, p14^ARF and p15^INK4b and the ANRIL gene which encodes a non-coding RNA. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis which causes CAD. We ascertained whether 9p21 genotype had an influence on CDKN2A/CDKN2B/ANRIL expression levels in VSMCs, VSMC proliferation and VSMC content in atherosclerotic plaques. Immunohistochemical examination showed that VSMCs in atherosclerotic lesions expressed p16^INK4a, p14^ARF and p15^INK4b. Analyses of primary cultures of VSMCs showed that the 9p21 risk genotype was associated with reduced expression of p16^INK4a, p15^INK4b and ANRIL (P = 1.2 × 10⁻⁵, 1.4 × 10⁻² and 3.1 × 10⁻⁹) and with increased VSMC proliferation (P = 1.6 × 10⁻²). Immunohistochemical analyses of atherosclerotic plaques revealed an association of the risk genotype with reduced p15^INK4b levels in VSMCs (P = 3.7 × 10⁻²) and higher VSMC content (P = 5.6 × 10⁻⁴) in plaques. The results of this study indicate that the 9p21 variation has an impact on CDKN2A and CDKN2B expression in VSMCs and influences VMSC proliferation, which likely represents an important mechanism for the association between this genetic locus and susceptibility to CAD.