Chemokine-like factor 1 (CKLF1) is a cytokine, which has a detrimental effect on the multiple disease progression. Our previous studies reported that arterial injury induced the upregulation of CKLF1 expression in artery at 7–14 days after injury. Here, using a rat carotid balloon injury model, we found that CKLF1 knockdown in the injured site abolished neointimal formation and even decreased medial area; contrarily, CKLF1 overexpression developed a thicker neointima than controls, demonstrating that CKLF1 exerted positive effects on neointimal hyperplasia and the accumulation of vascular smooth muscle cells (VSMC). The mechanism study indicated that CKLF1 reduced susceptibility to the cell cycle G2/M arrest and apoptosis, and thereby speeding up VSMC accumulation. This role of CKLF1 was tightly associated with phosphatidylinositol (PI) 3-kinase signaling pathway. CKLF1 increased the expression of four isoforms of the PI3-kinase catalytic subunits, which in turn activated its downstream targets Akt and an effector NF-κB accepted as critical transcription factors of cell survival and proliferation. Furthermore, RNA-sequencing analysis revealed that CKLF1 had wide-ranging roles in regulating the expression of genes that mainly engaged in cell apoptosis and innate immune response. Collectively, the data allow us to conclude that high level CKLF1 after artery injury switches the balance of VSMC proliferation and apoptosis through PI3K/AKT/NF-κB signaling and consequently leads to neointimal hyperplasia. The findings shed insight into new treatment strategies to limit restenosis based on CKLF1 as a future target.