Smooth muscle cells are called on to proliferate during vascular restructuring but must return to a nonproliferative state if remodeling is to appropriately terminate. To identify mediators of the reacquisition of replicative quiescence, we undertook gene expression screening in a uniquely plastic human vascular smooth muscle cell line. As proliferating VSMCs shifted to a contractile and nonproliferative state, expression of TIMP-3, Axl, and KIAA0098 decreased whereas expression of complement C1s, cathepsin B, cellular repressor of E1A-activated genes increased. Wilms’ tumor 1-associating protein (WTAP), a nuclear constituent of unknown function, was also upregulated as VSMCs became nonproliferative. Furthermore, WTAP in the intima of injured arteries was substantially upregulated in the late stages of repair. Introduction of WTAP complementary DNA into human VSMCs inhibited their proliferation, with a corresponding decrease in DNA synthesis and an increase in apoptosis. Knocking down endogenous WTAP increased VSMC proliferation, because of increased DNA synthesis and G₁/S phase transition, together with reduced apoptosis. WTAP was found to associate with the Wilms’ tumor-1 protein in human VSMCs and WTAP overexpression inhibited the binding of WT1 to an oligonucleotide containing a consensus WT1 binding site, whereas WTAP knockdown accentuated this interaction. Expression of the WT1 target genes, amphiregulin and Bcl2, was suppressed in WTAP-overexpressing VSMCs and increased in WTAP-deficient VSMCs. Moreover, exogenous amphiregulin rescued the antiproliferative effect of WTAP. These findings identify WTAP as a novel regulator of the cell cycle and cell survival and implicate a WTAP-WT1 axis as a novel pathway for controlling VSMC phenotype.