The dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) contribute to the formation of vascular lesions. In this study, the regulation of aortic carboxypeptidase-like protein (ACLP) expression in VSMCs was investigated. After mouse carotid injury, the expression of ACLP increases in the dedifferentiated VSMCs of the neointima in a pattern that differs from that of smooth muscle α-actin. To better understand the regulation of ACLP in VSMCs, we characterized the 21-exon mouse ACLP gene and 5′-flanking region and examined its promoter activity. In transient transfection assays, 2.5 kb of the ACLP 5′-flanking sequence directed high levels of luciferase reporter activity in primary cultured rat aortic smooth muscle cells, and this activity was not dependent on serum response factor. We identified a positive element between base pairs −156 and −122 by analysis of 5′ deletion and mutant constructs. By use of electrophoretic mobility shift assays with rat aortic smooth muscle cell nuclear extracts, Sp1 and Sp3 transcription factors bound to this region, and transfection assays in D.Mel.2 cells revealed that both Sp1 and Sp3 transactivated the ACLP promoter. Transgenic mice harboring the −2.5-kb ACLP promoter upstream from a nuclear-targeted LacZ gene were generated, and expression was detected in the VSMCs of large blood vessels, arterioles, and veins. Interestingly, ACLP promoter–LacZ reporter activity increased within the neointimal VSMCs of injured carotid vessels, consistent with the expression of the endogenous ACLP protein. The ACLP promoter may provide a novel tool to target gene expression to dedifferentiated VSMCs.