Recently, we have shown that a specific Rho-kinase inhibitor, Y27632 (R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide), prevents neointima formation after vascular injury associated with increased terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL)+ smooth muscle cells. Because the mechanism of the action of Y27632 remains unclear, we investigated the expression changes in Bcl family proteins, apoptosis regulators of smooth muscle cells, in the rat carotid artery after balloon injury (BI). Y27632 (BI + Y group) or saline (BI group) was administered peritoneally from Day 1 to Day 14 after BI. Y27632 markedly prevented neointima formation at Day 14. In the BI group, TUNEL+ smooth muscle cells were transiently increased in the neointima, but not in the media, with a peak at Day 7, returning to a lower level by Day 14. Y27632 significantly increased TUNEL+ smooth muscle cells at Days 7 and 14. Smooth muscle cell apoptosis was confirmed by electron microscopic examination. At Day 14, although proapoptotic Bax was slightly, but not significantly, increased in the BI group, it was significantly upregulated in the BI + Y group. Antiapoptotic Bcl-xL was upregulated in the BI group, and the upregulated Bcl-xL was not affected by Y27632. These findings indicate that Rho-kinase inhibition induces neointimal smooth muscle cell apoptosis through Bax upregulation, resulting in reduced neointima formation.