There is accumulating evidence that Ca²⁺-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, CaMKII, as a mediator of Ca²⁺ signals regulating VSM proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKII gene products while passaged primary cultures of de-differentiated VSM cells express primarily CaMKII₂, a splice variant of the gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKII₂ protein and significant decrease in CaMKII protein within 30 hrs, coinciding with onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKII₂ in primary cultured cells using siRNA resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKII₂ activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multi-nucleated cells. These results indicate that CaMKII₂ is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation.